U.S. patent application number 16/345612 was filed with the patent office on 2019-09-12 for stable water isotope labeling and magnetic resonance imaging for visualization of rapidly dividing cells.
The applicant listed for this patent is Nataliya BUXBAUM, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVIC, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVIC. Invention is credited to Nataliya BUXBAUM, Donald FARTHING, Ronald GRESS, Martin LIZAK, Natella MAGLAKELIDZE, Helmut MERKLE, Brittany OLIVER.
Application Number | 20190274616 16/345612 |
Document ID | / |
Family ID | 60321000 |
Filed Date | 2019-09-12 |
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United States Patent
Application |
20190274616 |
Kind Code |
A1 |
BUXBAUM; Nataliya ; et
al. |
September 12, 2019 |
STABLE WATER ISOTOPE LABELING AND MAGNETIC RESONANCE IMAGING FOR
VISUALIZATION OF RAPIDLY DIVIDING CELLS
Abstract
This disclosure generally relates to stable water isotope
labeling followed by detection via MRI (swiMRI), including
deuterium MRI (dMRI) and .sup.17O MRI, for visualizing rapidly
dividing immune cells within target and/or lymphoid organ/s and/or
tissues affected by chronic graft-versus-host disease (cGVHD).
Using deuterated water labeling, followed by dMRI, a distinction in
deuterium signal was detected in a target organ (e.g. liver) of the
cGVHD-affected mice compared to unaffected mice, i.e. syngeneic
HSCT recipient mice, where the host and donor are matched, and
normal (unmanipulated) mice.
Inventors: |
BUXBAUM; Nataliya;
(Bethesda, MD) ; FARTHING; Donald; (Bethesda,
MD) ; LIZAK; Martin; (Bethesda, MD) ; MERKLE;
Helmut; (Bethesda, MD) ; MAGLAKELIDZE; Natella;
(Bethesda, MD) ; OLIVER; Brittany; (Bethesda,
MD) ; GRESS; Ronald; (Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BUXBAUM; Nataliya
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY,
DEPARTMENT OF HEALTH AND HUMAN SERVIC |
Bethesda
Bethesda |
MD
MD |
US
US |
|
|
Family ID: |
60321000 |
Appl. No.: |
16/345612 |
Filed: |
October 27, 2017 |
PCT Filed: |
October 27, 2017 |
PCT NO: |
PCT/US2017/058856 |
371 Date: |
April 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62414554 |
Oct 28, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 2503/40 20130101;
G01R 33/3635 20130101; A61B 5/055 20130101; G01R 33/4828 20130101;
G01R 33/34069 20130101; G01R 33/34061 20130101; A61B 5/413
20130101; G01R 33/34092 20130101 |
International
Class: |
A61B 5/00 20060101
A61B005/00; A61B 5/055 20060101 A61B005/055; G01R 33/34 20060101
G01R033/34; G01R 33/36 20060101 G01R033/36; G01R 33/48 20060101
G01R033/48 |
Goverment Interests
GOVERNMENT INTEREST STATEMENT
[0002] The present subject matter was made with U.S. government
support. The U.S. government has certain rights in this subject
matter.
Claims
1. A method for NMR/MRI imaging to predict or detect an occurrence
of graft-versus-host disease in a subject; the method comprising:
providing a stable water isotope enriched fluid; where the stable
isotope enriched fluid is administered to the subject; allowing the
stable water isotope from the enriched fluid to incorporate into
rapidly dividing cells of the subject over a period of time;
determining the enrichment level of stable water isotope in a total
body water of the subject; wherein the enrichment level is
determined at least one of: before, during, or after the period of
time; positioning the subject within a magnetic field of an
energized NMR/MRI system, where the NMR/MRI system comprises a
probe tuned to measure a resonance frequency of the stable water
isotope and a resonance frequency of a proton (H); performing
magnetic resonance imaging (MRI) to detect a level of stable water
isotope enrichment in the rapidly dividing cells contained within
one or more organs or tissue of the subject; wherein the level of
stable water isotope enrichment in the rapidly dividing cells is
greater than a background enrichment of the stable water isotope in
the total body water of the subject; comparing the concentration of
the stable water isotope in the one or more organs or tissue of the
subject to a control subject, wherein the control subject does not
have graft-versus-host disease; and diagnosing the occurrence or a
likelihood of occurrence of graft-versus-host disease prior to or
during the clinical presentation of graft-versus-host disease
symptoms in the subject.
2. The method of claim 1 where the stable water isotope is
deuterium (.sup.2H), .sup.17O, or both.
3. The method of claim 1 where the total body water enrichment of
deuterium (.sup.2H), .sup.17O, or both is approximately 5% or
greater.
4. The method of claim 1 where the rapidly dividing cells comprise
one or more types of activated immune cells.
5. The method of claim 1 where the probe tuned to measure a
resonance frequency of the stable water isotope is also tuned to
measure a resonance frequency of a proton.
6. The method of claim 1, where the magnetic resonance further
comprises chemical shift imaging (CSI).
7. The method of claim 1, where the chemical shift imaging
comprises gathering images of a target organ or target tissue.
8. The method of claim 1, wherein the images of the target organ or
target tissue are correlated with anatomical (proton) imaging.
9. The method of claim 1, wherein administering the stable water
isotope labeled enriched fluid comprises injecting the enriched
fluid in the subject, providing the enriched fluid for ingestion,
or both.
10. A method for in vivo NMR/MRI imaging to detect rapidly dividing
cells in organs or tissues of subjects for diagnosis, monitoring,
detecting a medical condition; the method comprising: providing a
stable isotope enriched fluid; wherein the stable isotope enriched
fluid is enriched with deuterium (.sup.2H), .sup.17O, or
combinations thereof and where the stable isotope enriched fluid is
injected and/or ingested by the subject; allowing the deuterium
(.sup.2H) and/or .sup.17O from the enriched labeling fluid to
incorporate into rapidly dividing cells of the subject over a
labeling period; determining the enrichment level of stable water
isotope in a total body water of the subject before the labeling
period, during the labeling period, after the labeling period, or
combinations thereof; positioning the subject within a magnetic
field of an energized NMR/MRI system, where the NMR/MRI system
comprises a probe tuned to measure a resonance frequency of
deuterium (.sup.2H) and/or .sup.17O and tuned to measure a
resonance frequency of a proton (H) for anatomical imaging;
performing magnetic resonance imaging to detect stable water
isotope enrichment in the rapidly dividing cells within the organs
or tissue of the subject that is greater than a background
enrichment of the total body water of the subject; comparing the
concentration of deuterium enrichment in the organs and/or tissues
of the test subject with disease to the subject without the
condition AND detecting the occurrence or recurrence of the medical
condition based on the presence of the rapidly dividing cells prior
to or contemporaneous with a clinical presentation of the medical
condition.
11. The method of claim 10, where the accumulation of the rapidly
dividing cells in the subject indicates the presence of at least
one of a cancer, graft-versus-host disease, an immunological
disorder, or an infection.
12. The method of claim 10, wherein the stable isotope enriched
fluid is included in culture media or in a manufacturing process
for an immunotherapeutic product.
13. The method of claim 10, wherein the immunotherapeutic product
is labeled ex vivo prior to administration to the subject, then
imaged post administration to the subject to visualize and/or
monitor their localization within the subject.
14. The method of claim 10, wherein the medical condition comprises
cancer, an infection, or an immune disorder.
15. An NMR/MRI probe for use in a magnetic resonance imaging
system, the probe comprising: a first radiofrequency coil
comprising dual parallel rectangular loops, where the rectangular
loops each comprise a loop capacitor; and a second radiofrequency
coil comprising at a pair of double saddle coils; where each pair
of the double saddle coils is connected in series with an in-line
capacitor.
16. The probe of claim 15, where first radiofrequency coil is a
liner coil tuned to measure proton resonance.
17. The probe of claim 15, where the rectangular loops of the first
radiofrequency coil are connected in parallel to a transmission
line through a tune/match network comprising a tuning capacitor and
a matching capacitor.
18. The probe of claim 15, where the first radiofrequency coil is
terminated at an impedance of 50 ohms.
19. The probe of claim 15, where second radiofrequency coil is a
quadrature coil tuned to measure deuterium resonance.
20. The probe of claim 15, where each saddle coil has an arc of
approximately 120 degrees.
21-23. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/414,554, filed Oct. 28, 2016, which is herein
incorporated by reference in its entirety.
FIELD
[0003] The present disclosure relates to systems, methods, and
devices for the non-radioactive visualization of rapidly dividing
cells using stable water isotope labeling followed by detection via
stable isotope nuclear magnetic resonance imaging.
BACKGROUND
[0004] The identification and treatment of diseases and/or
conditions characterized by the presence of rapidly dividing cells
is often delayed by the need to perform a biopsy before making an
initial diagnosis. Performing a biopsy can be an invasive
procedure, particularly when tissue must be collected from internal
organs. By the time such a diagnosis is made, the disease or
condition has progressed. For example, chronic graft-versus-host
disease (cGVHD) is a prevalent and highly morbid condition
affecting allogeneic hematopoietic stem cell transplant
("allogeneic HSCT" or "AHSCT") recipients. Currently, there are no
diagnostic imaging features or validated biomarkers predictive of
impending or active cGVHD. It is known that biopsies performed on
HSCT recipients are risky and difficult, as these patients are
often on systemic immunosuppression.
[0005] Early detection of cGVHD and other diseases or conditions
characterized by the presence of rapidly dividing cells, such as
cancer or infection, would likely improve the long-term prognosis
of the patient. Non-invasive methods for such early detection are
desirable to limit the need for invasive biopsies only to confirm
initial findings and minimize additional physical impact on the
patient.
SUMMARY
[0006] The systems, methods, and devices of the present disclosure
generally relate to the early detection of chronic
graft-versus-host disease (cGVHD) and other diseases or conditions
characterized by the presence of rapidly dividing cells, such as,
but not limited to cancers, infections, and autoimmune diseases. In
one aspect, a method for NMR/MRI imaging to predict or detect an
occurrence of graft-versus-host disease in a subject includes
providing a stable water isotope enriched fluid and administering
the stable isotope enriched fluid to the subject. The method also
includes allowing the stable water isotope from the enriched fluid
to incorporate into rapidly dividing cells of the subject over a
period of time and determining the enrichement level of stable
water isotope in a total body water of the subject. The enrichment
level is determined at at least one of: before, during, or after
the period of time.
[0007] The method further includes positioning the subject within a
magnetic field of an energized NMR/MRI system. The NMR/MRI system
includes a probe tuned to measure a resonance frequency of the
stable water isotope and a resonance frequency of a proton (H). The
method also includes performing magnetic resonance imaging (MRI) to
detect a level of stable water isotope enrichment in the rapidly
dividing cells contained within one or more organs or tissue of the
subject; wherein the level of stable water isotope enrichment in
the rapidly dividing cells is greater than a background enrichment
of the stable water isotope in the total body water of the subject.
The method further involves comparing the concentration of the
stable water isotope in the one or more organs or tissue of the
subject to a control subject, where the control subject does not
have graft-versus-host disease; and, lastly, diagnosing the
occurrence or a likelihood of occurrence of graft-versus-host
disease prior to or during the clinical presentation of
graft-versus-host disease symptoms in the subject.
[0008] The present disclosure also relates to an NMR/MRI probe for
use in an magnetic resonance imaging system. In one aspect, the
NMR/MRI probe includes a first radiofrequency coil including dual
parallel rectangular loops. The rectangular loops each have a loop
capacitor. The probe also includes a second radiofrequency coil
comprising at a pair of double saddle coils; where each pair of the
double saddle coils is connected in series with an in-line
capacitor.
[0009] Additional objectives, advantages, and novel features will
be set forth in the description that follows or will become
apparent to those skilled in the art upon examination of the
drawings and detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A and 1B depict anatomical drawings of mice that
received hematopoietic stem cell transplantation (HSCT), according
to one embodiment;
[0011] FIG. 2A is a graph depicting clinical scores relating to
cGVHD symptoms for syngeneic HSCT recipients and allogeneic HSCT
recipients, according to one embodiment.
[0012] FIGS. 2B and C are photographs of mice at different time
points post HSCT that correspond to the clinical scores for
allogeneic HSCT recipients provided in FIG. 2A, according to one
embodiment;
[0013] FIG. 3 is a bar graph depicting the collective number of
CD4+ T effector memory (T.sub.EM) cells extracted from mouse organs
and tissues from each described cohort (graft, syngeneic HSCT
recipient, normal mouse, allogeneic HSCT recipient), according to
one embodiment;
[0014] FIG. 4 is a bar graph depicting the collective number of
CD4+ T naive (T.sub.N) cells extracted from mouse organs and
tissues from each described cohort (graft, syngeneic HSCT
recipient, normal mouse, allogeneic HSCT recipient), according to
one embodiment;
[0015] FIG. 5 is a graph depicting deuterium enrichment in normal
(unmanipulated) mice, syngeneic HSCT recipients, and allogeneic
HSCT recipients that developed cGVHD, according to one
embodiment;
[0016] FIG. 6 is a flow diagram depicting a method for labeling
mice with deuterium and determining the deuterium enrichment in
rapidly dividing cells in such mice, according to one
embodiment;
[0017] FIGS. 7A-7F include photographs of several prototypes of
deuterium probes, and a magnetic resonance imaging (MRI) machine,
according to embodiments disclosed herein;
[0018] FIGS. 8A and B are deuterium MRI (dMRI) images overlaid on
anatomical/proton MRI images, according to one embodiment;
[0019] FIGS. 8C and D are images of the deuterium spectroscopy
peaks that correspond to the regions of maximal signal intensity in
the dMRI images in FIGS. 8A and 8B, respectively, according to one
embodiment;
[0020] FIG. 9 is an illustration of an experimental schema for in
vivo deuterated water labeling followed by dMRI imaging of rapidly
dividing cells following HSCT, according to one embodiment;
[0021] FIG. 10 is a bar graph depicting averages of normalized
deuterium chemical shift imaging (CSI) signals detected in the
liver of multiple mice following syngeneic and allogeneic HSCT
according to one embodiment;
[0022] FIG. 11 is a bar graph depicting averages of normalized
deuterium chemical shift imaging (CSI) signals detected in
quadriceps muscle tissue, of multiple mice following syngeneic and
allogeneic HSCT, according to one embodiment;
[0023] FIG. 12 depicts an MRI scan and a spectroscopic deuterium
peak for a single mouse with the deuterium CSI signal overlaid on
an anatomical/proton MRI, according to one embodiment;
[0024] FIGS. 13A-13B includes graphs illustrating deuterium dosing
with resultant isotopic enrichment in DNA base pairs
(isotopologues), according to one embodiment;
[0025] FIG. 14 is a photograph of a spectroscopic deuterium signal
comparing signals from deuterium labeled mouse tumor cells and a
phantom solution containing un-enriched water, according to one
embodiment;
[0026] FIG. 15 is a schematic drawing of a single saddle coil of a
dual coil dMRI probe, according to one embodiment;
[0027] FIG. 16 is another depiction of the graph of 2A depicting
clinical scores relating to cGVHD symptoms for syngeneic HSCT
recipients and allogeneic HSCT recipients, according to one
embodiment;
[0028] FIGS. 17A-17B include flow cytometry data illustrating
lymphoid immune reconstitution (thymus and spleen) in allogeneic
recipients according to one embodiment;
[0029] FIG. 18 includes charts and graphs presenting spleen CD4+ T
cell numbers and compositions for normal mice, syngeneic, and
allogeneic HSCT recipients, according to one embodiment;
[0030] FIGS. 19A-19J depict histology slides of target organs for
syngeneic and allogeneic recipients, according to one
embodiment;
[0031] FIGS. 20A-20C include charts and graphs presenting liver
CD4+ T cell numbers and compositions for normal mice, syngeneic,
and allogeneic HSCT recipients, according to one embodiment;
[0032] FIGS. 21A-21D include charts and graphs presenting dermal T
cell numbers and compositions for normal mice, syngeneic, and
allogeneic HSCT recipients, according to one embodiment;
[0033] FIGS. 22A-22E include charts and graphs presenting small
intestine lamina propria (LP) and intraepithelial (IE) CD4+ T cell
numbers and compositions for normal mice, syngeneic, and allogeneic
HSCT recipients, according to one embodiment;
[0034] FIGS. 23A-23E include anatomical drawings and charts
presenting CD4+ T cell pool sizes, compositions and distributions
for normal mice, syngeneic and allogeneic HSCT recipients,
according to one embodiment;
[0035] FIGS. 24A-24D include charts presenting cell gain kinetics
for T.sub.Reg cells in the spleen and liver, according to one
embodiment;
[0036] FIGS. 25A-25D include charts and graphs presenting label
loss kinetics for T.sub.Reg cells in the liver appear to be driven
by increased propensity for apoptosis rather than trafficking,
according to one embodiment;
[0037] FIGS. 26A-26C include graphs presenting CD4.sup.+ T.sub.EM
in vivo cell kinetics, according to one embodiment;
[0038] FIGS. 27A-27E includes charts and graphs presenting cell
gain kinetics with concurrent rapid label loss in spleen and liver
in allogeneic recipients, according to one embodiment;
[0039] FIGS. 28A-28D include images and charts related to the in
vivo deuterium labeling followed by dMRI diagnosis of cGVHD in the
target organ, according to one embodiment; and
[0040] FIGS. 29A-29B includes schematic representations of
components of a (.sup.1H-.sup.2H) proton-deuterium coil, according
to one embodiment.
[0041] Reference characters indicate corresponding elements among
the view of the drawings. The headings used in the figures do not
limit the scope of the claims.
DETAILED DESCRIPTION
[0042] The present disclosure generally relates to the early
detection of chronic graft-versus-host disease (cGVHD) and other
diseases or conditions characterized by the presence of rapidly
dividing cells, such as, but not limited to cancers, infections,
and autoimmune diseases. In particular, the present disclosure
describes improved systems and methods for labeling whole
organisms, and/or organs, and/or cells with stable water isotopes,
including hydrogen and oxygen, (i.e. deuterium (.sup.2H) and heavy
oxygen (.sup.17O)) followed by magnetic resonance imaging for the
stable water isotope (e.g. deuterium MRI (dMRI) and/or .sup.17O
MRI), for visualization of rapidly dividing cells. The present
disclosure also relates to the use of deuterium and oxygen stable
water isotopes as labeling agents for imaging patients post
allogeneic hematopoietic stem cell transplantation ("allogeneic
HSCT" or "AHSCT") for diagnosing of early and/or ongoing cGVHD.
While the systems and methods are primarily disclosed and described
regarding the use of deuterium (.sup.2H), the systems and methods
may be configured for use with either deuterium (.sup.2H), heavy
oxygen (.sup.17O), or both. Other stable water isotopes may also be
used. Stable water isotope MRI (swiMRI), as used herein,
encompasses both, dMRI and .sup.17O MRI. In one aspect, swiMRI may
include the simultaneous imaging of deuterium and heavy oxygen. In
other aspects, the stable water isotopes may be imaged
separately.
[0043] Rapidly dividing cells, as used herein, may include, but are
not limited to, T cells and other immune cells that infiltrate
target organs affected by cGVHD. In the various embodiments
disclosed, swiMRI allows early detection of cGVHD in a
non-radioactive and inexpensive manner, and allows for the
diagnosis and monitoring of active or ongoing cGVHD. Alternatively,
as deuterium preferentially incorporates into rapidly dividing
cells, swiMRI may be used for non-invasive in vivo tumor imaging,
including visualization of neoplastic/cancer cells. The systems and
methods of the present disclosure offer inexpensive and
non-radioactive alternatives to positron emission tomography
(PET).
[0044] In another aspect, according to various embodiments
disclosed herein, swiMRI may allow visualization and localization
of immunotherapeutic products following infusion into an animal or
human subject. Examples of such products include chimeric antigen
receptor T cells (CAR T cells), tumor infiltrating lymphocytes
(TILs), and other adoptive immunotherapies, if such products
undergo stable water isotope labeling in culture (during
manufacture).
[0045] In another aspect, the present disclosure also relates to a
specially configured nuclear magnetic resonance (NMR)/magnetic
resonance imaging (MRI) coil or probe. In particular, the coil is
multi-tuned to detect signals from hydrogen, deuterium, and oxygen
isotopes, and combinations thereof simultaneously.
Pre-Clinical Modeling
[0046] A pre-clinical mouse model of cGVHD was used to
quantitatively measure in vivo kinetics of fluorescence-activated
cell sort (FACS) purified T cell subsets extracted from
cGVHD-affected organs. Some underlying principles of the model were
derived from studies using gas chromatography-tandem mass
spectrometry (GC-MS/MS) methods for measuring deuterium-labeled
deoxyadenosine in DNA extracted from T cell subsets, published as
Sensitive GC-MS/MS method to measure deuterium labeled
deoxyadenosine in DNA from limited mouse cell populations; by
Farthing, D. E. et al., Anal. Chem. 85, 4613-4620 (2013) which is
incorporated herein by reference in its entirety. Additional
underlying principles of the models were derived from studies
contained in the draft manuscript entitled "Just add water: T cell
subset kinetics and deuterium MRI of graft-versus-host disease"; by
Buxbaum, N. P. et al., which is and incorporated herein by
reference in its entirety from U.S. Provisional Application No.
62/414,554, filed Oct. 28, 2016.
[0047] In one aspect, the biology of cGVHD in the pre-clinical
model and similarly in patients is mediated by T cells.
Specifically, the genetic differences between host and donor drive
donor T cells to recognize host tissues as foreign, which results
in a multi-organ inflammatory disease in the host organs and
tissues--commonly referred to as graft-versus-host disease. While
cGVHD is mediated by T cells, the in vivo cell processes of T cell
division, death, and trafficking are not well characterized. In
clinical studies, peripheral blood is amenable to investigation,
but T cell populations found in the blood may not accurately
reflect T cell composition in target and/or lymphoid organs and
tissues. Therefore, the disclosed mouse model was useful in
evaluating T cells in blood, lymphoid, and target organs at
sequential biologically relevant time points following
transplantation. The relevant time points include pre-symptomatic
early events in cGVHD pathogenesis (day +14) to clinically apparent
cGVHD (day +28). A differential distribution of T cell subsets in
cGVHD (AHSCT, where the host and donor are purposefully mismatched
at minor immune antigens) versus immune reconstitution without
cGVHD (syngeneic hematopoietic stem cell transplant (HSCT), where
the host and donor are genetically identical) was identified.
[0048] FIGS. 1A-B are graphic representations of CD4+ T cell
distributions in the syngeneic HSCT setting (control) and
allogeneic HSCT (cGVHD affected) mice at day +28 following HSCT. In
particular, FIG. 1A depicts a mouse 100A that underwent syngeneic
(matched) HSCT, while FIG. 1B is a depiction of a mouse 100B that
underwent allogeneic (mismatched) HSCT and developed cGVHD. The
representations identify the organs and tissues that have the
highest number of CD4+ T cells in each cohort and the most
prevalent CD4+ T cell subset found in those organs and tissues. In
the syngeneic HSCT recipients (FIG. 1A), CD4+ T cells are primarily
of the naive phenotype and are found in lymphoid organs and
tissues, including but not limited to the spleen 102A, thymus 102B,
and lymph nodes 102C and 102D. In contrast, in the allogeneic HSCT
recipients (FIG. 1B), CD4+ T cells are primarily of the effector
memory phenotype and are found in the target organs and tissues,
including but not limited to the liver 104A, gastrointestinal tract
104B and 104C, and the skin 104D, but not in the lymphoid ones. Of
note, the CD4+T.sub.EM cells found in the target organs and tissues
104A-D of allogeneic HSCT recipients were found to have high DNA
deuterium enrichment levels (15-20%), when labeled to 5% total body
water (TBW) for days and up to weeks following infusion of an HSCT
graft.
[0049] Besides localization differences between syngeneic and
allogeneic HSCT recipients, the types of CD4+ T cells differ
between two cohorts. Specifically, the predominant phenotype of
CD4+T cells in the syngeneic recipient mice is T naive (T.sub.N);
while in the allogeneic recipient, the phenotype is primarily T
effector memory (T.sub.EM).
[0050] Data from the pre-clinical model of cGVHD to study T cell
populations in vivo is provided in FIGS. 1A-B and FIGS. 2A-C. FIG.
2A is a graph 200 depicting clinical scores relating to cGVHD
symptoms for syngeneic HSCT recipients 202 and allogeneic HSCT
recipients 204, according to one embodiment. The clinical scores
are determined based on presence of erythema (redness) and scaling
on the ears, tails, and paws, and surface area of skin affected by
alopecia (hair loss). The mice in the allogeneic group were driven
to cGVHD by minor deliberate mismatching between the donor and
host, while the mice in the syngeneic cohort were matched to the
donor. Each group was subject to a total body irradiation (TBI)
conditioning regimen performed one day before the infusion of the
transplant graft. FIGS. 2B and 2C are photographs of mice at
different time points post HSCT that correspond to the clinical
scores 204 in FIG. 2A.
[0051] The organs and compartments affected by cGVHD in the
pre-clinical mouse model were studied using flow cytometry,
immunohistochemistry (IHC), in vivo kinetics, or combinations
thereof, as described more fully below in the section under the
heading: An Analysis of T-cell kinetics and a Working Example of
T-cell Labeling and deuterium MRI imaging of cGVHD. In particular,
flow cytometry and IHC were used to observe cGVHD in the skin,
small intestine (i.e. duodenum, jejunum, ileum, and cecum), liver,
spleen, thymus, and lymph nodes. Peripheral blood was assessed by
flow cytometry. In vivo kinetics studies were performed on purified
CD4+ T cell subsets extracted from the spleen (lymphoid organ) and
liver (cGVHD target organ) of syngeneic and allogeneic HSCT
recipient mice.
[0052] Additional observations are shown in FIGS. 3 and 4. The
graph 300 of FIG. 3 depicts the collective number of CD4+ T
effector memory (T.sub.EM) cells extracted from mouse organs and
tissues from each described cohort (graft 302, syngeneic HSCT
recipient 304-306, normal mouse 308, and allogeneic HSCT recipient
310-312. In one aspect, the graft 302 is made from bone marrow and
spleen of the donor and the number of CD4+T.sub.EM cells in the
graft was determined by flow cytometry.
[0053] For the normal (untransplanted) mice, syngeneic HSCT
recipients, and allogeneic HSCT recipients, CD4+T.sub.EM cells were
extracted from the whole spleen, the whole thymus, the small bowel
(lamina propria and intraepithelial layer), the whole liver, the
skin and peripheral blood. The total number of skin-resident
CD4+T.sub.EM cells was determined in by counting the number of
CD4+T.sub.EM cells in a 1 cm.sup.2 sample and determining the total
body surface area based on weight. Similarly, the number of
CD4+T.sub.EM cells in a blood sample volume and the total blood
volume based on weight were used to calculate the total number of
CD4+T.sub.EM cells in circulation.
[0054] The graph 400 of FIG. 4 depicts the collective number of
CD4+ T naive (T.sub.N) cells extracted from mouse organs and
tissues from each described cohort (graft 402, syngeneic HSCT
recipient 404-406, normal mouse 408, allogeneic HSCT recipient
410-412). In one aspect, the graft 402 is made from bone marrow and
spleen of the donor and the number of CD4+ T.sub.N cells in the
graft was determined by flow cytometry. For the normal
(untransplanted) mice, syngeneic HSCT recipients, and allogeneic
HSCT recipients, CD4+ T.sub.N cells were extracted from whole
spleen, whole thymus, small bowel (lamina propria and
intraepithelial layer), whole liver, skin and peripheral blood. The
total number of skin-resident CD4+ T.sub.N cells was determined in
by counting the number of CD4+ T.sub.N cells in a 1 cm.sup.2 sample
and determining the total body surface area based on weight.
Similarly, the number of CD4+ T.sub.N cells in a blood sample
volume and the total blood volume based on weight were used to
calculate the total number of CD4+ T.sub.N cells in
circulation.
[0055] As shown, the number of CD4+T.sub.EM cells is higher in the
allogeneic HSCT cohort compared to the other cohorts, as shown FIG.
3. The number of CD4+ T naive (T.sub.N) cells increases over the
course of syngeneic immune reconstitution, while the number of
T.sub.N cells in the allogeneic HSCT remains low, as shown in FIG.
4. Concurrently, a differential distribution of CD4+ T cell subsets
in cGVHD-affected animals compared to unaffected mice is observed,
as shown in FIGS. 1A-B.
Deuterated Water Labeling
[0056] According to various embodiments, deuterium was provided for
uptake into rapidly dividing cells for subsequent detection via
deuterium chemical shift imaging (CSI), a type of magnetic
resonance imaging, also referred to herein as dMRI. Quantitative
measurements of in vivo T cell kinetics in lymphoid and target
organs were obtained by applying deuterated water labeling and
de-labeling post HSCT, followed by extraction and purification of T
cell subsets from target and lymphoid organs, then quantitatively
measuring deuterium labeled and unlabeled fractions of
deoxyadenosine (DNA base pair). Fraction of newly divided cells was
then mathematically determined.
[0057] In one aspect, deuterium labeling was achieved by
intraperitoneal injection of 100% .sup.2H.sub.2O with NaCl added to
make this fluid isotonic, then maintaining the achieved 5% .sup.2H
enrichment in total body water (TBW) by providing 8% .sup.2H.sub.2O
in drinking water to mice. Deuterium within total body water
diffuses rapidly into all tissues and is excreted unchanged by the
kidneys. Therefore, according to various embodiments, deuterium
labeled fluids may be provided to an animal or human subject via
injected and/or ingested fluid yielding a deuterium concentration
of about 5% TBW. In various other embodiments, higher enrichments
of the deuterium labeling up to and including approximately 20% TBW
have been safely achieved. Higher TBW enrichments (up to 20% TBW
.sup.2H) may improve sensitivity of dMRI detection. Such
enrichments should be safe, as deuterium is non-radioactive,
tasteless, colorless and without known side effects at these
doses.
[0058] The provided deuterium incorporates into the DNA of dividing
cells constitutively through the de novo nucleoside synthesis
pathway, as described in Measurement of cell proliferation by heavy
water labeling; by Busch, R. et. al., Nat Protoc 2, 3045-3057
(2007). High enrichment of deuterium (15-20%) in cellular DNA of
rapidly diving cells, i.e. CD4+ T effector memory cells (Tem) in
cGVHD target organ (liver), was measured in the disclosed cGVHD
mouse model following TBW labeling to 5% for 7 days by measuring
.sup.2H labeled and unlabeled dA in cellular DNA using gas
chromatography and tandem mass spectrometry (GC-MS/MS), as shown in
FIG. 5.
[0059] FIG. 5 is a graph 500 depicting deuterium enrichment in
deoxyadenosine (dA), a DNA base pair, of CD4+ T cells, either
T.sub.N 502, 506, and 510 or T.sub.EM 504, 508, 512, extracted from
liver parenchyma of normal (unmanipulated) mice, syngeneic HSCT
recipients, and allogeneic HSCT recipients that developed cGVHD,
respectively. For each cohort, the enrichment in dA was measured
via GC-MS/MS following 1 week of in vivo deuterium labeling to 5%
in total body water. In one aspect, varied labeling and de-labeling
periods may measure deuterium gain and loss from cellular DNA via
GC-MS/MS.
[0060] FIG. 13 includes graphs 1300 and 1302 of .sup.2H.sub.2O dose
and isotopic enrichments in DNA base pairs, such as dA
isotopologues. As shown, increasing the .sup.2H.sub.2O level in TBW
(or culture media) results in nearly linear increases in deuterium
incorporation into DNA base pairs.
[0061] In one aspect, an advantage of using stable water isotopes,
such as deuterated water (.sup.2H.sub.2O), over other methods of
cell division measurements, such bromodeoxyuridine (BrdU),
tritiated thymidine, or carboxyfluorescein succinimidyl ester
(CFSE), is that deuterium is non-radioactive, safe, and non-toxic
at doses up to 20% in total body water. Deuterium does not affect
rates of DNA synthesis and no ex vivo labeling is required.
[0062] FIG. 6 is a flow chart depicting one embodiment of a method
for determining the deuterium enrichment in DNA of rapidly dividing
cells in mice after labeling. As shown, the method includes the
steps of providing .sup.2H.sub.2O to a mouse via intraperitoneal
injection, then in the drinking water, followed by excision of the
organ of interest to isolate the desired cell population by
fluorescence-activated cell sorting (FACS). Next, DNA is extracted,
hydrolyzed to release deoxyadenosine (dA), and then purified using
a C-18 column. The purified DNA is derivatized by methylation.
Fractions of the labeled and unlabeled dA are then measured using
GC-MS/MS, an example of such enrichment in dA is shown in FIG. 5.
Lastly, once the level of deuterium enrichment is measured, the
fraction of the newly divided cells is calculated.
Deuterium Magnetic Resonance Imaging Probe
[0063] In various embodiments, a specially configured multi-tuned
deuterium probe or coil is used to gather a signal from the
deuterium labeled cells, an organ (e.g. liver), or an organism,
after in vivo labeling with deuterium. In one particular
embodiment, the probe is a quadrature (orthogonal) deuterium probe
that provides an improved signal to noise ratio over earlier
deuterium probes.
[0064] The disclosed deuterium probe has high sensitivity in the
deuterium channel and can also obtain images in the hydrogen
(proton) channel. Preferably, the proton coil and the deuterium
coil create homogeneous radiofrequency fields (B1) within the
target volume. FIG. 7 includes photographs (A)-(E) of various
embodiments of the multi-tunable dMRI deuterium probe 700 for use
with a suitable magnet, including but not limited to the 9.4 Tesla
Bruker magnet, shown in FIG. 7(F).
[0065] One embodiment of the probe 700 is partially shown in FIG.
15. The probe 800 includes a quadrature deuterium coil 702 and a
linear proton coil (not shown) rotated by 45 degrees. The proton
coil structure is a "quasi Helmholtz pair" of two parallel
rectangular loops with integrated capacitors. The loops are
connected in parallel and terminated at the transmission line with
an impedance of 50.OMEGA. using a balanced tune/match network.
[0066] The deuterium coil is composed of two crossed pairs of
double saddle coils 702 of 120-degree arc, with each pair connected
in series with a single in-line capacitor 704 each. The saddles 702
of the (i) channel and the (q) channel are arranged orthogonally
and are isolated to typically less than 0.2% residual coupling. The
deuterium coil is also matched and tuned, using a tune/match
network 706 and is terminated at a 50.OMEGA. impedance transmission
line 708.
[0067] In various other embodiments, the deuterium probe is
substantially similar to the coil disclosed in U.S. Pat. No.
5,323,113, issued on Jun. 21, 1994 to Cory, et al., which is
incorporated herein by reference in its entirety. The present
probe, however, differs over the probe of U.S. Pat. No. 5,323,113,
in that the deuterium probe may be tuned to detect signals from
proton, deuterium, and other stable isotopes simultaneously.
[0068] Various other embodiments, or portions thereof, for the
multi-tuned probe 700, are shown in FIGS. 29A-B. FIG. 29A (a) is a
schematic of the "quasi Helmholtz" .sup.1H (proton)
transmit/receive coil 2900 for scout (anatomical) imaging. The
arrow 2902 indicates the B.sub.1 direction in regard to the magnet
Cartesian coordinates (B.sub.0=Z=horizontal). FIG. 29A (b) is a
schematic of the (q)-channel of the "quasi Helmholtz" saddle-type
.sup.2H transmit/receive coil 2904 for chemical shift imaging
(CSI). The double arrow 2906 indicates the B.sub.1 direction in
regard to the magnet Cartesian coordinates (B.sub.0=Z=horizontal).
FIG. 29A (c) is a schematic of the (i)-channel of the "quasi
Helmholtz" saddle-type .sup.2H transmit/receive coil 2908 for CSI.
The double arrow 2910 indicates the B.sub.1 direction in regard to
the magnet Cartesian coordinates (B.sub.0=Z=horizontal). FIG. 29B
is a schematic of the setup for .sup.1H and .sup.2H imaging at 9.4
Tesla magnetic flux density. All coils are mounted on a cylindrical
former. The 1H (proton) transmit/receive coil 2900, is connected to
the MRI system, including the transmit/receive switch 2912. As
shown in FIG. 29B, the deuterium coil pairs 2904 and coil 2908 are
positioned at a 90.degree. angle. The orthogonal arrangement and
90.degree. phase delayed feeding of the radio frequency ("RF")
current reduces the power requirement for a well-defined flip angle
to 1/2, compared to that needed for a single-saddle coil. In
addition, the signal-to-noise ratio is increased by a factor of 2.
A power transmitter 2920 at .sup.2H frequency is connected at port
#1 2912 of the hybrid via a band pass filter 2922 to a quadrature
hybrid for power splitting and phase creating. Ports #2 2914 and #3
2916 of the hybrid are connected to the two .sup.2H coils 2904 and
2908 using identical length cables. The combined signal is received
at port #4 2918 of the hybrid that acts as a transmit/receive
switch. The signal is passed to another band pass filter 2924 to
the x-receiver port 2926.
[0069] Using dMRI in CSI mode and a single sagittal 8-mm slice
covering the thoracic and abdominal cavities, a significant
difference in in vivo liver deuterium signal in the cGVHD-affected
mouse compared to control mice, syngeneic HSCT recipient and normal
(unmanipulated) mouse, was detected. In one embodiment, the probe
700, such as the probe pictured in FIG. 7(D) provided greater
sensitivity for deuterium detection, and allowed acquisition of
chemical shift images (CSI) with improved spacial resolution and/or
shorter imaging times. In this embodiment, the slice thickness was
reduced to approximately 3 mm Higher levels of deuterium CSI were
measured using dMRI in the livers of mice affected by cGVHD at day
28 as compared to control syngeneic HSCT livers at day 28 post
transplant. Besides CSI deuterium imaging, the current probe shown
in FIG. 7(D), allows for regular proton imaging. Anatomical images
may be captured, onto which deuterium CSI signals can be overlaid.
This enables the calculation of deuterium CSI signal coming
specifically from liver and spleen and not from the surrounding
tissues/spaces. The probe pictured in FIG. 7(D) is being used for
cGVHD imaging of mice.
[0070] FIGS. 8A-D illustrate example data obtained using a dMRI
probe, such as those shown in FIGS. 7A-D and FIG. 15 to perform in
vivo dMRI imaging of a syngeneic HSCT recipient and an allogeneic
HSCT recipient (cGVHD-affected mouse), following deuterated water
labeling for 21 days (day +7 to +28 post HSCT). FIGS. 8A and 8B
shows dMRI images 800 and 802 overlaid on anatomical/proton MRI
images 804 and 806. The images were obtained at day +28 from mice
following syngeneic (A) and allogeneic (B) HSCT and deuterated
water labeling to approximately 5% total body water for 21 days
(i.e., day +7 to +28). It was observed at day +28, that the
allogeneic HSCT recipient mice have clinical signs and symptoms of
cGVHD. FIG. 8A is an image of a syngeneic HSCT recipient mouse,
while FIG. 8B is an image of a cGVHD-affected mouse. Both mice were
imaged with a 9.4 T Bruker magnet, similar to that shown in shown
in FIG. 7(F). The images of FIGS. 8A and 8B were generated as 8-mm
sagittal slices covering the lower thoracic and peritoneal
cavities.
[0071] FIGS. 8C and 8D are images of the deuterium spectroscopy
peaks that correspond to the regions of maximal signal intensity in
the liver depicted on the dMRI images in FIGS. 8A and 8B,
respectively. The deuterium peak 808 from the liver of allogeneic
HSCT recipient mouse, as shown in FIG. 8D, is higher than the peak
810 of the syngeneic HSCT recipient mouse, as shown in FIG. 8C.
[0072] FIG. 9 is an illustration for one embodiment of a scheme 900
for labeling and imaging rapidly dividing cells. As shown, the
method begins with total body irradiation (TBI) conditioning
performed one day prior to the infusion of a transplant graft (day
-1), indicated as 902. In the embodiment shown, the bone marrow
transplant (BMT) graft, containing bone marrow stem cells and T
cells from the donor spleen, is infused on day 0, indicated as 904.
One week after the transplant, indicated as 906, the subjects
receive an intraperitoneal (IP) bolus of normal saline made in 100%
.sup.2H.sub.2O, and thereafter receive drinking water containing 8%
(.sup.2H.sub.2O) deuterated water. In one embodiment, the labeling
schema resulted in approximately 5% TBW enrichment in the
subjects.
[0073] At various intervals after the labeling, chemical shift
dMRI, .sup.17O MRI, or swiMRI images of the subjects are captured.
In one embodiment, the chemical shift dMRI images are gathered at
weekly intervals, day +14, day +21, and day +28, as indicated by
908, 910, and 912 respectively. In various other embodiments,
different intervals for imaging after labeling may be used.
According to one aspect, a 5% .sup.2H.sub.2O phantom is also
scanned simultaneously with the subject to provide reference data.
The phantom may be included in one or more scans 908-912. FIG. 14
is a photograph of a spectroscopic deuterium signal 1402 from
cultured mouse tumor cells having a dA deuterium enrichment of
.about.50%, and a signal 1404 from a phantom solution containing
un-enriched water (0.015% .sup.2H.sub.2O in H.sub.2O (v/v)).
[0074] In various embodiments, the slice thickness of the captured
dMRI images may be varied. In one preferred aspect, thinner slices
(e.g. less than or equal to 3-mm are captured to improve image
resolution), as illustrated in FIGS. 10-11, but a summation of data
from several slices may be necessary to better estimate total organ
deuterium and/or .sup.17O CSI signal.
[0075] FIG. 10 is a bar graph 1000 presenting averages of
normalized deuterium chemical shift imaging (CSI) signals detected
in liver (target organ of cGVHD) of multiple mice following
syngeneic 1002 and allogeneic 1004 HSCT, with error bars
representing standard error, and p-values 1006. Three syngeneic and
four allogeneic HSCT recipients were imaged and three 3-mm slices
were obtained per animal. The liver deuterium CSI signals for each
mouse were normalized to a deuterium CSI signal detected from a
phantom of 5% .sup.2H.sub.2O in H.sub.2O (v/v), that was imaged
concurrently with each animal. All signals were measured on day +28
following HSCT and after 21 days of deuterated water labeling (day
+7 to +28) to 5% total body water.
[0076] FIG. 11 is bar graph 1100 presenting averages of normalized
deuterium chemical shift imaging (CSI) signals detected in
quadriceps muscle tissue, which is not affected by cGVHD. The
signals were gathered from multiple mice following syngeneic 1102
and allogeneic 1104 HSCT, with error bars representing standard
error, and p-values 1106. Three syngeneic and four allogeneic HSCT
recipients were imaged and three 3-mm slices were obtained per
animal. The muscle deuterium CSI signals for each mouse were
normalized to a deuterium CSI signal detected from a phantom of 5%
.sup.2H.sub.2O in H.sub.2O (v/v), that was imaged concurrently with
each animal. The signals were measured on day +28 following HSCT
and after 21 days of deuterated water labeling (day +7 to +28) to
5% total body water.
[0077] After capturing data using any of the embodiments for
labeling and imaging disclosed, a cGVHD-affected liver was readily
distinguishable from a syngeneic control liver, in vivo, after an
average of 21 days of continuous labeling to maintain deuterium
enrichment of 5% in TBW. In various aspects, a distinction between
a cGVHD-afflicted affected liver and a syngeneic control liver may
be observed in as little as 14 days.
[0078] For example, FIG. 12 shows captured deuterium chemical shift
ionization (CSI) data 1200 overlaid on an anatomical/proton MRI
image 1202. The MRI scan 1204, which includes the CSI data 1200 and
the anatomical image 1202, is shown with a spectroscopic deuterium
peak 1206. The data were gathered on day +28 post allogeneic HSCT,
for a single mouse. The deuterium CSI signal and anatomical/proton
MRI image were obtained simultaneously on a Bruker 9.4 T magnet,
such as that shown in FIG. 7(E), using a multi-tuned probe, such as
that shown in FIG. 7(D). The mouse was labeled according to the
methods disclosed with deuterated water to approximately 5%
.sup.2H.sub.2O in total body water for 21 days (day +7 to +28).
Three approximately 3-mm thick coronal slices through the
mid-abdomen were captured. The liver and spleen were identified
through anatomical imaging, and the deuterium CSI signals for each
slice through the liver and spleen are shown.
[0079] In various aspects, the supplementation of deuterium and
other stable water isotopes can range from days to months, to
maintain the desired TBW enrichment. Once deuterium supplementation
is discontinued, TBW deuterium enrichment will return to baseline
(0.015%) in approximately 7 to 14 days in mice and people,
respectively.
In Vivo Imaging with .sup.17O
[0080] In various embodiments, systems and methods to visualize
rapidly dividing cells in vivo may use .sup.17O as a
non-radioactive label. .sup.17O is a stable isotope having
gyromagnetic properties amenable to MRI. As a gas, .sup.17O has
been used clinically to label autologous red blood cells (RBCs) for
vascular MRI imaging. In this aspect, red blood cells may be
extracted from a patient and passed through a chamber of gaseous
.sup.17O. .sup.17O is loaded onto hemoglobin molecules inside RBCs,
which then are returned to the patient's circulation and function
as a contrast agent allowing visualization of blood vessels.
Alternatively, concentrations of gaseous .sup.17O may be used to
measure oxygen metabolism to detect ischemia.
[0081] Various embodiments of the systems and methods disclosed may
use .sup.17O in a liquid form as isotopic water having the formula
H.sub.2.sup.17O. Similar to deuterium contained in .sup.2H.sub.2O,
the .sup.17O isotopic water would be injected and/or ingested and
would incorporate into the DNA of rapidly dividing cells as a
labeling agent. The de-labeling kinetics of .sup.17O are
substantially similar to those of deuterium enrichment as
previously described.
[0082] Stable isotopic water containing deuterium and .sup.17O
(i.e. .sup.2H.sub.2.sup.17O) may be used to "double" label rapidly
dividing cells and enhance swiMRI signal for the early detection
and monitoring of cGVHD, and other conditions characterized by
rapidly dividing cells. In embodiments using H.sub.2.sup.17O or
.sup.2H.sub.2.sup.17O, one or more swiMRI probes similar to those
shown in FIG. 7 may be used. Similarly, the MRI probes would be
multi-tuned to capture signals from protons for anatomical imaging,
and deuterium (.sup.2H), and isotopic oxygen (s.a. .sup.17O) for
spectroscopic imaging, simultaneously.
[0083] Besides using stable isotopes of hydrogen and/or oxygen in
water as labels for detecting cGVHD, .sup.2H and .sup.17O may also
be used for non-invasive and non-radioactive in vivo imaging of
tumors as an alternative to positron emission tomography (PET). As
shown in FIG. 14, a clear spectroscopic deuterium signal is
obtained from mouse tumor cells labeled in vitro with deuterated
water during culture.
[0084] Besides in vivo labeling followed by imaging, various other
embodiments of the systems and methods disclosed may be used for ex
vivo labeling followed by in vivo or in vitro dMRI imaging. By way
of example and not limitation, immunotherapy products may be
labeled ex vivo during production. Subsequent deuterium (.sup.2H),
and isotopic oxygen (s.a. .sup.17O) swiMRI can visualize the in
vivo localization of the infused cells.
[0085] Total body water (TBW) enrichment with stable water isotopes
of hydrogen and/or oxygen (.sup.2H.sub.2O, H.sub.2.sup.17O, or
.sup.2H.sub.2.sup.17O) can be measured in small volumes of body
fluid, such as saliva, urine or blood. A novel method for testing
TBW is described in a draft manuscript by Farthing, D F, et al.
"Uncharted Waters--Comparing stable isotopic forms of heavy water
incorporation into DNA of proliferating cells," which is found in
U.S. Provisional Application No. 62/414,554, filed Oct. 28, 2016
and which is incorporated herein by reference in its entirety. Such
measurements may be useful to monitor animal and human subject
compliance with label intake, some of which may occur in the
outpatient setting (unmonitored). Total body water (background)
stable water isotope enrichment has and would be measured prior to
swiMRI scan and/or various intervals before, during, and after the
labeling period. Measurement of TBW enrichment with stable water
isotopes is important because higher than 0.015% deuterium
concentration and 0.04% .sup.17O concentration found in regular
water are necessary to generate high enrichment of label into
rapidly dividing cells facilitating subsequent detection with
swiMRI.
An Analysis of T-Cell Kinetics and a Working Example of T-Cell
Labeling and Deuterium MRI Imaging of cGVHD
[0086] T cells are central to the biology of chronic
graft-versus-host disease (cGVHD), a morbid and prevalent
allo-immune complication of hematopoietic stem cell transplantation
(HSCT). Using in vivo deuterated water labeling in a mouse model of
GVHD, we measured kinetics of CD4+ T cell subsets, i.e. T
regulatory (T.sub.Reg), T effector memory (T.sub.EM), and T naive
(T.sub.N), in lymphoid and target organs. We found that a low
(<<1) T.sub.Reg to CD4+ T.sub.EM ratio rather than T.sub.Reg
to T.sub.CON, both in circulation and systemically, is predictive
of impending GVHD and established disease. Despite high
proliferation in lymphoid and target organs, the systemic T.sub.Reg
number is low due to reduced T.sub.Reg survival in target organs.
These findings, in part, underlie the limited efficacy of treatment
regimens for GVHD that inhibit general T cell proliferation,
without targeting particular subsets. By identifying contrasting
distribution of CD4+ T cell subsets in a target organ (e.g. liver)
of diseased animals with their differential deuterium DNA
enrichment, we developed a novel deuterium magnetic resonance
imaging (dMRI) approach to discern GVHD-affected animals from the
control HSCT recipients. We show that deuterated water labeling as
used for kinetics studies followed by dMRI can facilitate a
non-invasive and non-radioactive in vivo diagnosis of GVHD.
[0087] While T cells are at the biological forefront of diseases
across disciplines, such as autoimmunity, infectious disease,
inherited and acquired immunodeficiency, malignancy, and
transplantation, their in vivo behavior, encompassing generation of
new cells, cell survival, and trafficking, is difficult to measure
and interpret. Further complexity arises from T cells comprising of
subsets, which differ not only by phenotype, but also by function.
Allogeneic hematopoietic stem cell transplantation (AHSCT) is a
treatment strategy widely used to cure malignant and non-malignant
diseases. Chronic GVHD (cGVHD) is a morbid, prevalent, and
refractory AHSCT barrier, characterized by systemic immune
dysregulation driven by allo-reactive donor T cells. T regulatory
(T.sub.Reg) cells play a critical role in cGVHD, with several
animal and clinical studies demonstrating the potential of
T.sub.Reg cells to treat this disease. The origin of the imbalance
between regulatory and allo-reactive T cells in cGVHD should be
explored further, and understanding of in vivo T cell subset
kinetics should illuminate the biology that underlies the
imbalance.
[0088] Deuterium labeling (via water or glucose) for measuring in
vivo cell kinetics has been extensively used for over a decade. It
provides an alternative to other nucleoside analogs, such as
bromodeoxyuridine (BrdU) and tritiated thymidine (.sup.3HTdR) that
are typically not applicable to clinical studies due to toxicity,
and incorporate into the DNA of dividing cells via the salvage
pathway of nucleotide synthesis, which is unpredictable and varies
by cell type. Indeed, T cell subsets differ in their dependence on
this pathway based on their stage of maturation (i.e. naive versus
memory). In contrast, deuterium is incorporated into cellular DNA
through the constitutive de novo nucleotide synthesis pathway,
which is not subject to regulation. Since stable isotopes are not
radioactive, deuterium labeling lends itself to clinical
translation with relatively small amounts of deuterium enrichment
(.about.5%) in total body water required for kinetics measurements.
Pioneering in vivo kinetics studies were conducted in patients with
HIV, which measured T cells (CD4+ and CD8+), then many other cell
types and conditions. To date, studies involving stable isotopes
measured T cell kinetics in circulation, a dynamic cellular
compartment, while systemic cell half-lives were mathematically
estimated. The logistical complication of extracting cells from
organs and tissues in patients precluded measurement of systemic
cellular dynamics, but egress from circulation and migration into
tissue is key to T cell function. These limitations are abrogated
in small animal studies since cells of interest can be extracted
from a number of organs. However, to our knowledge, no previous
studies have employed deuterium labeling to measure kinetics of T
cells or other cell types in multiple compartments. We show that
the same CD4+ T cell subset (i.e. T.sub.Reg) can have vastly
different kinetics in lymphoid versus target organs. It is
desirable to evaluate biologically distinct subsets of T cells (not
simply CD4+ versus CD8+ T cells), because we found that CD4+ naive,
memory, and regulatory cells have differential in vivo
kinetics.
[0089] GVHD has been defined by an imbalance between
immunoregulatory and pathogenic CD4+ T cells. While our work
confirms these findings, we provide further insight and detailed
understanding of the immune imbalance by defining it as a low
T.sub.Reg to CD4+ T.sub.EM ratio (<<1), and provide
measurements of T cell subset behaviors that underlie the low
ratio. The T.sub.Reg to T conventional (T.sub.CON) or T.sub.Reg to
CD4+ T.sub.CON ratios prominently figure in discussions of
post-HSCT immunity and T.sub.Reg studies, but, in our model, may
not distinguish cGVHD-affected from unaffected cohorts, in
circulation, lymphoid or target organs. These ratios are not
currently used to predict ongoing or impending cGVHD in patients.
In contrast, the ratio we focus on, T.sub.Reg to CD4+ T.sub.EM, not
only defines which animals are affected by cGVHD, but also predicts
cGVHD in advance of disease manifestations. The T.sub.Reg to CD4+
T.sub.EM ratio may be altered in patients with GVHD, and should be
explored further. Finally, our novel use of deuterium labeling to
facilitate non-invasive and non-radioactive magnetic resonance
imaging (MRI) of cGVHD may introduce an objective criterion for
cGVHD diagnosis, which is currently challenging and primarily
subjective.
[0090] Results
[0091] CD4+ T cell immune reconstitution in cGVHD is skewed toward
distribution to target rather than lymphoid organs, with a
predominance of the T.sub.EM phenotype. As previously described,
allogeneic graft recipients consistently developed cGVHD by
post-transplant day 28; while at day +14 clinical scores were not
consistent with cGVHD. (FIG. 16).
[0092] Lymphoid atrophy and low absolute CD4+ T cell numbers in the
thymi and lymph nodes were observed in allogeneic recipients at day
+14 and persisted through day +28. In addition, thymi of allogeneic
recipients contained a mature (CD44 high) CD4+ T cell infiltrate
with a marked reduction in T cell precursors (FIG. 17A). At day
+14, splenomegaly was seen in allogeneic recipients. It was
characterized by mild extra medullary hematopoiesis and a high
number of CD4+T cells, primarily of the T.sub.EM (CD4+ FoxP3- CD44
high CCR7-) phenotype (FIGS. 18A-E). Following initial size
increase, the spleen underwent atrophy with a near absence of CD4+
T cells in the spleen at day+28 (FIGS. 18A-D). In congenic
allogeneic HSCT experiments, CD4+ T cells harvested from the host
thymus and spleen at various times following transplantation, day
+7 through +35, were of a donor-derived peripherally expanded
mature phenotype (Thy1.1 Ly 9.2 CD44 high) (FIG. 17B).
[0093] The following caption applies to FIG. 16: "Clinical scoring
data for mice undergoing HSCT." (a) Typical clinical score curve
for mice in syngeneic and allogeneic cohorts at specified post HSCT
time points. The Green threshold line represents a clinical score
of 0.6, minimum score for considering mice as having clinical
evidence of cGVHD. Data for day 0 through +35 are representative of
more than ten independent experiments.
[0094] The following caption applies to FIG. 17: "Lymphoid immune
reconstitution in allogeneic recipients is characterized by donor
peripherally-expanded mature CD4+ T cell predominance" (a) Thymic
immune-reconstitution following HSCT evaluated by flow cytometry at
day +30. (i) Syngeneic recipients show a predominance of double
positive thymocytes (CD4.sup.+CD8.sup.+). (ii) Allogeneic
recipients show a predominance of single positive CD4.sup.+, and
near absence of double positive and double negative thymocytes
(CD4.sup.-CD8.sup.-). (iii) These single positive CD4.sup.+ cells
are primarily of memory (CD44.sup.+) phenotype. (iv) Congenic
markers showed that CD4.sup.+CD44.sup.+ cells were of donor-derived
peripherally expanded phenotype (Ly9.1.sup.-Thy1.2.sup.-). Data are
representative of three independent experiments (i, ii) and two
independent experiments (iii, iv). (b) Flow cytometry gating
strategy for spleen samples, with (i) syngeneic and (ii) allogeneic
cohort representative data for day +14.
[0095] The following caption applies to FIG. 18: "Spleen CD4+ T
cell number and composition for normal mice, syngeneic, and
allogeneic HSCT recipients." (a and b) The mean total CD4.sup.+ T
cell number for syngeneic day +14=1.7.times.10.sup.6, syngeneic day
+28=4.1.times.10.sup.6, allogeneic day +14=2.9.times.10.sup.6,
allogeneic day +28=0.2.times.10.sup.6, normal=10.9.times.10.sup.6.
The size of each pie was normalized to one. Data shown in (b)
represent the mean, with SEM error bars. (c) The mean spleen
T.sub.EM cell number in allogeneic versus syngeneic recipients
measured at day +14 and +28, with SEM error bars. Data for four
independent experiments were pooled for these analyses (n=2 to 7
mice per cohort per time point) and are representative of more than
ten independent experiments. **, P<0.01."
[0096] The target organs affected by cGVHD, i.e. integument, liver,
and small intestine were characterized by lymphocytic infiltrates
(FIGS. 19A-J). Liver parenchyma of allogeneic recipients contained
approximately a log higher absolute number of CD4+ T cells by day
+14 compared to the syngeneic counterparts (FIGS. 20A-C). Similar
to other target organs affected by cGVHD, the predominant CD4+ T
cell subset was T.sub.EM, and a decreased T.sub.Reg
(CD4+FoxP3+CD25+): T.sub.EM ratio was observed (FIG. 20A, C) Dermal
parenchyma sections of the allogeneic cohort had higher CD4+
absolute counts than syngeneic counterparts (FIGS. 21A-B).
Furthermore, T.sub.EM phenotype predominated in the dermal CD4+ T
cell pool in the allogeneic setting both at day +14 and day +28
(FIG. 21D). In the small intestine, CD4+ T cell number was
increased in the lamina propria (LP) and intraepithelial (IE)
compartment of allogeneic compared to syngeneic recipients (FIGS.
22A, C). Furthermore, the T.sub.EM proportion and total number were
significantly higher in allogeneic LP than syngeneic and normal LP
by day +28, and in the IE compartment at both time points (FIGS.
22B, D).
[0097] The following caption applies to FIG. 19: "Mouse target
organ histology and flow cytometry characteristics for syngeneic
and allogeneic HSCT recipients." Target organ histology for
syngeneic and allogeneic recipients. (a) Representative syngeneic
HSCT recipient skin section, 200.times.; normal skin histology is
observed at day +14 and day +28. (b) Allogeneic HSCT recipient skin
section, 200.times., day +28. Prominent hyperkeratosis and
acanthosis are observed. While the magnification and orientation of
the section are similar for (a) and (b), in (b) hyperkeratosis
precludes visualization of additional skin layers (subcutis and
muscularis not visualized). (c) Syngeneic skin epithelial section,
600.times.; normal histological appearance. (d) Allogeneic skin
epithelial section, 600.times., day +28; intraepithelial
lymphocytes evident with dyskeratotic epithelial cells. (e)
Syngeneic skin hair follicle section, 600.times.; normal
histological appearance. (f) Allogeneic skin hair follicle section,
600.times., day +28; intraepithelial peri-follicular lymphocytes
and dyskeratotic epithelial cells are evident. (g) Syngeneic liver
section, 400.times., day +14; normal histologic appearance. (h)
Allogeneic liver section, 400.times., day +14; moderate lymphocytic
periductal infiltrate is present. (i) Syngeneic small intestine
section, 400.times., day +14; normal intestinal crypt and villi
histology is observed. (j) Allogeneic section, 400.times., day +14;
crypt hyperplasia, increased number of epithelial cells, and a
lymphocytic infiltrate are observed. Histology findings are
representative of two independent experiments (n=5 mice per cohort
per time point).
[0098] The following caption applies to FIG. 20: "Parenchymal liver
CD4.sup.+ T cell number and composition for normal mice, syngeneic,
and allogeneic HSCT recipients." (a and b) The mean total CD4.sup.+
T cell number for syngeneic day +14=0.2.times.10.sup.6, syngeneic
day +28=0.4.times.10.sup.6, allogeneic day +14=1.7.times.10.sup.6,
allogeneic day +28=1.1.times.10.sup.6, normal=0.5.times.10.sup.6.
The size of each pie was normalized to one. Data shown in (b)
represent the mean, with SEM error bars. (c) T.sub.Reg: T.sub.EM
ratio is consistently lower in the allogeneic setting compared to
that of syngeneic and normal cohorts. Data shown represent the
mean, with SEM error bars. Data for two independent experiments
were pooled for this analysis (n=5 to 8 mice per cohort per time
point), and are representative of three independent experiments for
day +14. *, P<0.05; **, P<0.01.
[0099] The following caption applies to FIG. 21: "Dermal T cell
number and composition for normal mice, syngeneic and allogeneic
HSCT recipients." (a) The average total number of dermal T cells
for syngeneic day +14=0.6.times.10.sup.6, syngeneic day
+28=1.6.times.10.sup.6, allogeneic day +14=1.5.times.10.sup.6,
allogeneic day +28=3.7.times.10.sup.6, normal=2.4.times.10.sup.6.
The size of each pie was normalized to one. (b) Dermal CD4+ T cell
number for each experimental cohort measured at day +14 and +28.
(c) Representative flank dermal flow cytometry data for the
allogeneic cohort at day +28. Data for two independent experiments
were pooled, and are representative of three independent
experiments. (d) Dermal CD4.sup.+ T.sub.EM cell number for each
experimental cohort measured at day +14 and +28. * P=0.02, 0.04,
0.04, left to right; ** P=0.007.
[0100] The following caption applies to FIG. 22: "Small intestine
lamina propria (LP) and intraepithelial (IE) T cell number and
composition for normal mice, syngeneic and allogeneic HSCT
recipients." (a) Total number of small intestine LP CD4.sup.+ T
cells for each experimental cohort measured at day +14 and +28. *
P=0.04; ** P=0.002, 0.006, 0.002, left to right (b) Total number of
small intestine LP CD4.sup.+ T.sub.EM cells for each experimental
cohort measured at day +14 and +28. ** P=0.006. (c) Total number of
small intestine IE CD4.sub.+ T cells for each experimental cohort
measured at day +14 and +28. (d) Total number of small intestine IE
CD4.sup.+ T.sub.EM cells for each experimental cohort measured at
day +14 and +28. (e) Flow cytometry gating strategy for small
intestine LP samples is displayed, with the upper panels
representative of syngeneic data for day +14, and the lower panels
representative of allogeneic cohort data for day +14. Data for
three independent experiments were pooled for the analysis.
[0101] Overall, CD4+ T cell immune reconstitution in the syngeneic
transplant setting mirrored distribution and composition of T cells
in normal mice, i.e. lymphoid organs (FIG. 23A). The predominant
phenotype for CD4+ T cells in normal mice and syngeneic recipients
was naive (T.sub.N, Foxp3- CD44 low CCR7+) (FIGS. 23A-B). In the
syngeneic cohort, few CD4+ T cells were found outside of the
lymphoid tissues. In contrast, lymphoid organs in the allogeneic
setting underwent atrophy with few CD4+ T cells present in these
anatomic locations. Additionally, in the allogeneic cohort CD4+ T
cells were primarily localized to the target tissues, such as the
integument, liver, and gastrointestinal tract, and were mostly of
the T.sub.EM phenotype (FIGS. 23A, C). This resulted in a very low
(<<1) target organ, systemic, and peripheral blood T.sub.Reg
to T.sub.EM) ratio in the allogeneic setting (FIG. 20C and FIGS.
23(D-E). In contrast, the T.sub.Reg to T.sub.EM ratio was
significantly higher (>1) in the syngeneic and normal cohorts in
all the same sites (FIG. 20C and FIGS. 23D-E). To elucidate why
T.sub.Reg cells were diminished while T.sub.EM cells were expanded
in the allogeneic setting we obtained measurements of in vivo cell
kinetics for CD4+ T cell subsets.
[0102] The following caption applies to FIG. 23: "The CD4.sup.+ T
cell pool size, composition and distribution for normal mice,
syngeneic and allogeneic HSCT recipients." Cell numbers on display
in this set of figures were generated by adding CD4.sup.+ T cell
subset numbers extracted from whole spleen, whole parenchymal
liver, small intestine (intra-epithelial and lamina propria
layers), flank skin (calculated by adjusting excised section
numbers for calculated body surface area), and peripheral blood
(collected volume adjusted for calculated total blood volume. (e)
T.sub.R). (a) Graphic representation of the predominant CD4.sup.+ T
cell subset distribution in the syngeneic and allogeneic HSCT
recipients at day +28. CD4.sup.+ T cells were primarily found in
lymphoid organs and were of T.sub.N phenotype in the syngeneic
cohort. Pattern of CD4.sup.+ T cell distribution for the normal and
syngeneic cohorts was identical. In contrast, CD4.sup.+ T cells
were primarily found in target tissues (skin, liver, gut) with
predominance of T.sub.EM phenotype in allogeneic setting. (b) The
mean total T naive cell number measured within the graft, normal
mice, and syngeneic and allogeneic recipients at day +14 and +28,
with SEM error bars. (c) The mean total T.sub.EM and T.sub.Reg cell
numbers measured within the graft, normal mice, and syngeneic and
allogeneic recipients at day +14 and +28, with SEM error bars. (d)
T.sub.Reg: T.sub.EM ratio was >1 in the graft, syngeneic HSCT
recipients and normal mice, while it was <1 in cGVHD setting.
(e) T.sub.Reg: CD4+ T.sub.con ratio vs. T.sub.Reg: CD4+ T.sub.EM
ratio for normal mice, and syngeneic and allogeneic recipients at
d+28 in peripheral blood. Data shown represent the mean, with SEM
error bars. Data are representative of more than three independent
experiments. *, P<0.05; **, P<0.01.
[0103] In Vivo T.sub.Reg Kinetics Indicate Marked Proliferation in
Lymphoid and Target Organs and Diminished Survival in Target
Organs
[0104] T.sub.Reg number did not increase substantially over the
course of allogeneic immune reconstitution, while there was a
significant net increase following syngeneic transplantation (FIG.
23C). Meanwhile, cell gain kinetics for T.sub.Reg cells extracted
from the spleen and liver parenchyma showed high rates of label
gain for allogeneic and syngeneic cohorts (FIGS. 24A-B).
Furthermore, in the allogeneic setting T.sub.Reg cells experienced
label gain rates similar to those of T.sub.EM cells (FIGS. 24C-D).
While both subsets had similarly high rates of label gain in
lymphoid and target organs this did not result in an increased
systemic T.sub.Reg cell number, while the T.sub.EM cells were
abundant (FIG. 23D). This difference in absolute numbers despite
similar label gain kinetics could be driven by differential cell
loss kinetics. Indeed, our kinetics data suggest that T.sub.Reg
cell loss from the liver exceeds that of T.sub.EM subset (FIG.
25A). Furthermore, the key difference between the allogeneic liver
T.sub.Reg and the other CD4+ subsets was the presence of Caspase 3
staining (FIG. 25B), which indicated greater propensity for
apoptosis. When first-order elimination kinetics were applied to
the observed label loss rates, T.sub.Reg subset half-life in liver
was estimated to be roughly one-half of the T.sub.EM half-life.
Increased loss of T.sub.Reg cells in the target tissues may explain
why T.sub.Reg cells as a subset did not expand following HSCT in
the allogeneic recipients despite their high rates of label gain.
T.sub.Reg cells were not observed in target organs nor in
circulation in an appreciable number throughout the course of
allogeneic immune reconstitution, while T.sub.EM cells were
circulating and predominated in the target organs (FIGS. 23A, C and
FIGS. 25C-D).
[0105] The following caption applies to FIG. 24: "Cell gain
kinetics for T.sub.Reg cells in the spleen and liver." (a)
Percentage of new cells within the spleen-derived T.sub.Reg subset
for each experimental cohort measured at day +14 and +28, following
7 days of label administration for each time point. Data for five
independent experiments were pooled for the day +7 to +14 analysis
(each independent experiment represents a pooled sample from n=2 to
7 mice), and three independent experiments were pooled for day +21
to +28 analysis. (b) Percentage of new cells within the
liver-derived T.sub.Reg subset for each experimental cohort
measured at day +14 and +28, following 7 days of label
administration for each time point. Data for three independent
experiments were pooled for the day +7 to +14 analysis (each
independent experiment represents a pooled sample from n=5 to 8
mice), and two independent experiments for day +21 to +28 analysis.
(c) Percentage of new cells within the spleen-derived allogeneic
CD4+ subsets (T.sub.Reg, T.sub.EM, and T.sub.N) measured at day +14
and +28, following 7 days of label administration for each time
point. Data for five independent experiments were pooled for the
day +7 to +14 analysis, and three independent experiments were
pooled for day +21 to +28 analysis. (d) Percentage of new cells
within the liver-derived allogeneic CD4+ subsets (T.sub.Reg,
T.sub.EM and T.sub.N) measured at day +14 and +28, following 7 days
of label administration for each time point. Data for three
independent experiments were pooled for the day +7 to +14 analysis,
and two independent experiments for day +21 to +28 analysis. Data
shown represent the mean, with SEM error bars. *, P<0.05; **,
P<0.01.
[0106] The following caption applies to FIG. 25: "Label loss
kinetics for T.sub.Reg cells in the liver appear to be driven by
increased propensity for apoptosis rather than trafficking." (a)
Label loss kinetics for allogeneic liver-derived T.sub.Reg and
T.sub.EM cells. Data are representative of three independent
experiments for day +14 (n=5 to 8 mice per experiment per cohort),
day +28 represents n=5 to 8 mice. (b) Caspase 3 staining for
CD4.sup.+ T.sub.Reg, T.sub.EM, and T.sub.N subsets extracted from
the spleen and liver at day +14 (n=5 mice per cohort). (c) The mean
total T.sub.Reg cell number measured in the peripheral blood of
each experimental cohort at day +14 and +28, with SEM error bars.
(d) The mean total T.sub.EM cell number measured in the peripheral
blood of each experimental cohort at day +14 and +28, with SEM
error bars. Data are representative of four independent experiments
(n=2 to 6 mice per experiment per cohort). **, P<0.01.
[0107] In Vivo T.sub.EM Kinetics Indicate Marked Expansion in
Lymphoid and Target Organs and Suggest Trafficking to Target
Organs
[0108] In allogeneic recipients, the total number of T.sub.EM cells
in all evaluated tissues combined, i.e. spleen, liver, small
intestine, skin, peripheral blood, and lymph nodes approached 4
million by day +14, while only 0.06 million were contained in the
graft (FIG. 23C). This represents more than a 60-fold expansion of
this compartment. In comparison, in the syngeneic recipients, the
T.sub.EM compartment expanded roughly 10-fold (FIG. 23C). In
allogeneic recipients, label gain for the T.sub.EM subset in spleen
and liver was robust through day +14 (FIGS. 26A-B), and paralleled
the overall concurrent increase in the T.sub.EM cell number during
this period in both organs (FIGS. 18A, C and FIGS. 20A, B)).
Additionally, the T.sub.EM subset in both organs did not show
significant Caspase 3 staining (FIG. 25B). Label loss in the
T.sub.EM liver subset was slower than that of T.sub.Reg cells (FIG.
25(a)); while, in the spleen, T.sub.EM cells appeared to undergo
rapid label loss (FIG. 26C). A possible mechanism for T.sub.EM
subset label and absolute number loss from the spleen may be
trafficking. This was supported by the measurement of high T.sub.EM
number in circulation of allogeneic recipients at day +14 (FIG.
25(d)). T.sub.EM in vivo cell kinetics showed high label gain in
lymphoid and target organs, rapid loss from the lymphoid organ
concurrent with a high circulating number and target organ
infiltration, without increased propensity for apoptosis. Robust
expansion of the T.sub.EM subset in the allogeneic setting, both in
absolute number and with regard to prominent label gain, was
further investigated.
[0109] The following caption applies to FIG. 26: "CD4.sup.+
T.sub.EM in vivo cell kinetics show robust cell gain in the spleen
and liver, with rapid concurrent label loss kinetics in the spleen
and increased number in circulation." (a) Percentage of new cells
within the spleen-derived T.sub.EM subset for each experimental
cohort measured at day +14 and +28, following 7 days of label
administration for each time point. Data for five independent
experiments were pooled for the day +7 to +14 analysis (each
independent experiment represents a pooled sample from n=2 to 7
mice), and three independent experiments for day +21 to +28
analysis. (b) Percentage of new cells within the liver-derived
T.sub.EM subset for each experimental cohort measured at day +14
and +28, following 7 days of label administration for each time
point. Data for three independent experiments were pooled for the
day +7 to +14 analysis (each independent experiment represents a
pooled sample from n=5 to 8 mice), and two independent experiments
for day +21 to +28 analysis. (c) Label loss kinetics for
spleen-derived T.sub.EM cells of allogeneic versus syngeneic
recipients. Data are representative of two independent experiments
(each independent experiment represents a pooled sample from n=2 to
7 mice). Data are representative of four independent experiments
(n=2 to 6 mice per experiment per cohort). *, P<0.05; **,
P<0.01.
[0110] T.sub.N Expansion and Conversion to T.sub.EM Contributes to
T.sub.EM Predominance in Lymphoid and Target Tissues
[0111] CD4+ T.sub.N subset displayed high rates of label gain early
post transplantation in both syngeneic and allogeneic setting, with
the latter demonstrating higher rates in both lymphoid and target
tissues (FIGS. 27A-B). However, in contrast to the syngeneic graft
that reconstituted the host with a growing number of T.sub.N cells,
the allogeneic recipients had fewer T.sub.N cells than contained in
the graft at all points following transplantation (FIG. 23B). Label
loss for T.sub.N cells was appreciably faster in the spleen and
liver of allogeneic versus syngeneic recipients (FIGS. 27(C-D).
Meanwhile, Caspase 3 expression in T.sub.N cells was not increased
in either organ (FIG. 25B). The T.sub.N cells were decreased in
circulation of allogeneic recipients compared to the syngeneic
cohort and those of normal mice (FIG. 27E). Thus, rapid expansion
coupled with rapid loss potentially represents T.sub.N conversion
to the T.sub.EM phenotype. Incorporation of deuterium label into
DNA of a particular cellular subset occurs through cell division,
because deuterium label incorporates into DNA base pairs only
during transcription of new strands. Phenotypic conversion, i.e.
from T.sub.N to T.sub.Em, involves cell proliferation and
differentiation. Our label gain kinetics and flow cytometry data
suggest that most of the conversion to the T.sub.EM phenotype
occurs before day +14 in allogeneic recipients.
[0112] T.sub.N cells were not found in circulation of allogeneic
cohort at the evaluated time points following transplantation,
while T.sub.EM cells were found in circulation at day +14 (FIG. 27E
and FIG. 25D, respectively). This indicates that a likely site of
T.sub.N to T.sub.EM conversion is the spleen, with subsequent
egress of T.sub.EM cells from the spleen. In the syngeneic setting,
T.sub.N cells appeared to undergo high rates of label gain and
minimal label loss in both spleen and liver (FIGS. 27A-D). This
paralleled a growing absolute number of T.sub.N cells observed over
the course of syngeneic engraftment (FIG. 23B). T.sub.N expansion
was observed even in thymectomized syngeneic HSCT recipients,
suggesting lack of T.sub.N conversion to T.sub.EM is a key
difference between allogeneic and syngeneic immune reconstitution.
As expected, in normal mice little label gain or loss was measured
in the T.sub.N subset (FIGS. 27A-D), likely due to immune
homeostasis. To summarize, our data indicate that in the syngeneic
setting T.sub.N cells proliferate to expand the T.sub.N subset,
while in the allogeneic setting they proliferate and convert to
T.sub.EM.
[0113] The following caption applies to FIG. 27: "In allogeneic
recipients CD4.sup.+ T.sub.N cells display robust cell gain
kinetics with concurrent rapid label loss in spleen and liver, but
low circulating number." (a) Percentage of new cells within the
spleen-derived T.sub.N subset for each experimental cohort measured
at day +14 and +28, following 7 days of label administration for
each time point. Data for five independent experiments were pooled
for day +7 to +14 (each independent experiment represents a pooled
sample from n=2 to 7 mice), and three independent experiments for
day +21 to +28 analyses. (b) Percentage of new cells within the
liver-derived T.sub.N subset for each experimental cohort measured
at day +14 and +28, following 7 days of label administration for
each time point. Data for three independent experiments were pooled
for day +7 to +14 (each independent experiment represents a pooled
sample from n=5 to 8 mice), and two independent experiments for day
+21 to +28 analyses. (c) Label loss kinetics for spleen-derived
T.sub.N cells of allogeneic versus syngeneic recipients. Data are
representative of two independent experiments (each independent
experiment represents a pooled sample from n=2 to 7 mice). (d)
Label loss kinetics for liver-derived T.sub.N cells of allogeneic
versus syngeneic recipients. Data are representative of three
independent experiments for day +14 (n=5 to 8 mice per experiment
per cohort), day +28 represents n=5 to 8 mice. (e) The mean total
T.sub.N cell number measure in the peripheral blood for each
experimental cohort at day +14 and +28, with SEM error bars. Data
are representative of four independent experiments (n=2 to 6 mice
per experiment per cohort). *, P<0.05; **, P<0.01.
[0114] Deuterated Water Labeling Followed by In Vivo Deuterium
Magnetic Resonance Imaging (dMRI) Distinguishes Mice with from
Those without cGVHD
[0115] Our measurements of high deuterium (.sup.2H) enrichment in
deoxyadenosine (dA) of liver-infiltrating T.sub.EM cells (FIG. 28A)
and the high number of this cell type within target organs of
cGVHD-affected mice (FIGS. 20A and 23A, 23C), prompted us to
measure in vivo .sup.2H signal within organs of mice in both
cohorts. Based on the difference in .sup.2H levels between
background total body water enrichment (TBW) of .about.5% achieved
for kinetics studies and the resultant higher enrichment
(.about.15-20%) in the liver-infiltrating T.sub.EM subset, we
initiated deuterium (.sup.1H) chemical shift imaging (CSI), a type
of magnetic resonance imaging (MRI), in our mouse model. A custom
manufactured .sup.1H-.sup.2H coil for 9.4 Tesla magnet was used to
conduct mouse imaging (FIGS. 29A-29B). The detected .sup.2H signal
within livers of cGVHD-affected mice was significantly greater than
that measured in syngeneic controls at day +28, following 21-days
of .sup.2H.sub.2O labeling to TBW of .about.5% (FIG. 28(a-b)).
Furthermore, no difference in .sup.2H signal was observed within a
tissue not targeted by cGVHD in our mouse model, such as the
quadriceps muscle group (FIG. 28D)
[0116] The following caption applies to FIG. 28: "In vivo deuterium
labeling followed by dMRI facilitates diagnosis of cGVHD in the
target organ." (a) Deuterium chemical shift imaging (CSI) overlaid
on proton (anatomical) magnetic resonance imaging (MRI) obtained
with Bruker 9.7 T magnet on day +28 following AHSCT, after 21 days
(day +7 to +28) of .sup.2H.sub.2O labeling to total body water of
5%. For each mouse, 3 coronal slices 3-mm in thickness were
obtained through the mid-abdomen (region covering liver and
spleen); 5% .sup.2H.sub.2O phantom was imaged with each mouse to
provide a reference CSI signal. The MRI image obtained was rotated
180.degree. for the sake of convention. (b) DNA enrichment with
deuterium, measured in deoxyadenosine (dA) via GC-MS/MS for the
liver CD4+ T.sub.EM subset in the three cohorts at day +14. The
mean and SEM are displayed. *, P<0.5, ***, P<0.001. (c)
Average slice normalized deuterium CSI (nCSI) in the liver was
significantly higher in cGVHD-affected mice compared to syngeneic
recipients, while the unaffected tissue (muscle) deuterium nCSI did
not differ between cohorts, shown in (d). For (c) and (d), mean
nCSI is presented with SEM error bars, Data are representative of
two independent experiments (for the experiment shown, n=4 and 3
for allo and syn, respectively). ***, P<0.001.
[0117] The following caption applies to FIGS. 29A-B: "Components of
the (.sup.1H-.sup.2H) proton-deuterium coil." (a) Schematic of the
"quasi Helmholtz" .sup.1H transmit/receive coil for scout
(anatomical) imaging. The blue double arrow indicates the B.sub.1
direction in regard to the magnet Cartesian coordinates
(B.sub.0=Z=horizontal). (b) Schematic of the (q)-channel of the
"quasi Helmholtz" saddle-type .sup.2H transmit/receive coil for
chemical shift imaging (CSI). The red double arrow indicates the
B.sub.1 direction in regard to the magnet Cartesian coordinates
(B.sub.0=Z=horizontal). (c) Schematic of the (i)-channel of the
"quasi Helmholtz" saddle-type .sup.2H transmit/receive coil for
CSI. The green double arrow indicates the B.sub.1 direction in
regard to the magnet Cartesian coordinates (B.sub.0=Z=horizontal).
(d) Schematic of the setup for .sup.1H and .sup.2H imaging at 9.4
Tesla magnetic flux density. All coils are mounted on a cylindrical
former. The coil described in (a), shown here as a blue circle, is
connected in a standard way to the MRI system, including the
transmit/receive switch. The deuterium coil consists of two
identical dual saddle-shaped "quasi Helmholtz" pairs at a
90.degree. angle, described in (b) and (c) and shown here as red
and green circles, respectively. The orthogonal arrangement and
90.degree. phase delayed feeding of the RF current reduces the
power requirement for a well-defined flip angle to 1/2, compared to
that needed for a single-saddle coil. In addition, the
signal-to-noise ratio is increased by a factor of 2. A power
transmitter at .sup.2H frequency is connected at port #1 of the
hybrid via a band pass filter to a quadrature hybrid for power
splitting and phase creating. Ports #2 and #3 of the hybrid are
connected to the two .sup.2H coil ports using identical length
cables. The combined signal is received at port #4 of the hybrid
that acts as a transmit/receive switch. The signal is passed to
another band pass filter to the x-receive port.
[0118] Discussion:
[0119] Our work elucidates differential in vivo kinetics of CD4+ T
cell subsets, which lead to skewed immune reconstitution following
allogeneic hematopoietic stem cell transplantation (HSCT). This
pre-clinical model mirrors matched unrelated donor HSCT in patients
because minor antigen mismatch between donor and host induces
clinical GVHD. This model rapidly evolves into chronic GVHD with
short latency, which differs from patients in whom manifestations
are temporally separate from graft infusion and acute GVHD, however
the spectrum of clinical manifestations in patients is
recapitulated, for instance sclerotic dermal involvement, which is
pathognomonic for cGVHD is prominent. We show that subsets of
non-T.sub.Reg CD4+ T cells, which are often treated as a single
cell population and referred to as CD4+ T conventional (T.sub.CON),
have differential in vivo behavior. Quantitative dynamic in vivo
cell kinetics measurements have not been previously obtained for
subsets of non-T.sub.Reg CD4+ T cells. Additionally, we measured
these processes in multiple anatomic compartments. Previous studies
employed mathematical modeling to hypothesize in vivo cell kinetics
outside of the circulation while measuring deuterium label gain and
loss in circulating cells. In contrast, we obtained quantitative
measurements of deuterium gain and loss in T cell subsets from
lymphoid and target organs, and found the same subset (i.e.
T.sub.Reg) to behave differently depending on site. Bioluminescent
studies in mice have demonstrated differential trafficking of T
cell subsets in acute GVHD, however this methodology is based on
enumeration of cells in organs, which we also accomplished by
direct extraction from tissues and subset phenotyping by flow
cytometry. Enumeration would be unable to address cell kinetics,
i.e. whether minimal T cell subset accumulation in a particular
compartment (e.g. T.sub.Reg in liver) is due to diminished
proliferation or results from robust proliferation followed by
apoptosis; furthermore, T.sub.Reg as a subset were not compared to
T.sub.EM. Other studies evaluated lymphoid organs, but lymphoid
manifestations of GVHD do not provide a full view of in vivo
biology. For instance, in our cGVHD model, we show that lymphoid
and target organs are different with respect to T cell number,
subset proportions, and cell kinetics.
[0120] By direct lymphocyte extraction from tissue parenchyma, we
were able to map CD4+ T cell subset distribution to the relevant
targets of cGVHD, including liver, skin, and gastrointestinal
tract, the latter two have not been examined in the cGVHD setting
with regard to CD4+ T cell subsets. Similar to patients with cGVHD,
the allogeneic recipients in our model were lymphopenic when
peripheral blood was evaluated. Low CD4+ T cell numbers were also
found in the thymus, lymph nodes, and spleen; however, these
animals had a greatly increased number of CD4+ T cells in the
target organs, particularly of the T.sub.EM phenotype. A
system-wide imbalance between immune regulation and activation was
highlighted by the reduced ratio of T.sub.Reg to CD4+ T.sub.EM
cells in the setting of cGVHD compared to immune reconstitution
without antigenic mismatch. These findings indicate that
measurement of this ratio prospectively on patients undergoing HSCT
may represent an early biomarker of cGVHD. We show that the
T.sub.Reg to CD4+ T.sub.EM ratio<<1 (in any of the
compartments we evaluated, including blood) is the key parameter
that predicts which animals go on to develop cGVHD and consistently
identifies animals with clinically apparent cGVHD. This ratio is
superior to the T.sub.Reg to T.sub.CON ratio and the T.sub.Reg to
CD4+ T.sub.CON ratio, which are in wide use in HSCT and other
fields, as they are not altered across all relevant tissues
(lymphoid, target, and circulation) nor are they currently being
used in clinical assessments or scoring of GVHD in patients. This
highlights that evaluation of subsets of CD4+ T.sub.CON (i.e.
T.sub.EM) is biologically relevant, especially because CD4+
T.sub.CON subsets do not uniformly have similar in vivo behavior or
function.
[0121] Previous investigations indicated that differential T cell
proliferation was responsible for the imbalance between
immunoregulatory and pro-inflammatory elements observed in cGVHD.
However, we show that CD4+ T.sub.EM cells and T.sub.Reg cells
undergo robust expansion in lymphoid and target tissues. T.sub.Reg
number remains low, however, secondary to diminished survival in
the target organ of cGVHD. T.sub.EM in vivo kinetics pattern is one
of high label gain coupled with minimal concurrent label loss.
Therapy for GVHD has aimed to reduce general T cell proliferation,
while our findings indicate that both, immunoregulatory and
pathogenic, CD4+ T cell subsets expand robustly following
allogeneic HSCT. The T.sub.Reg subset is likely compromised by
increased propensity for apoptosis, rather than failure to
proliferate. This indicates that therapeutic interventions should
aim to reduce T.sub.EM expansion, avoid harming T.sub.Reg
proliferation, and promote T.sub.Reg survival. There have been
several promising examples of the latter approach for acute and
chronic GVHD, where T.sub.Reg survival was improved either through
manipulation of the cytokine milieu, for example low dose IL-2
therapy, or through affecting inherent changes in the T.sub.Reg
cells.
[0122] To target T.sub.EM subset expansion, it is important to
understand the dynamic cell kinetics of this subset over the course
of cGVHD. Our selective congenic graft experiments show that high
label gain in this subset within lymphoid and target organs is in
part driven by conversion of non-T.sub.Reg CD4+ T naive (T.sub.N)
cells to T.sub.EM phenotype. Conversion of T.sub.N to T.sub.EM
occurs early post-HSCT and is likely triggered by recognition of
allo-antigens, which does not occur in syngeneic recipients. The
conversion heralds impending cGVHD and is further perpetuated over
the course of cGVHD. As such, our data concur with others in that
T.sub.N cells contained in the graft are critical to acute and
chronic GVHD induction. In our study, donor CD4+ T.sub.N cells
undergo conversion shortly after graft infusion, and the effector
subset they convert to then mediates cGVHD in host organs. A key
point that reconciles previously published studies and ours is that
for cGVHD initiation the priming for conversion of T.sub.N to
T.sub.EM has to occur in the host, as T.sub.EM (FoxP3-) selected
grafts do not result in cGVHD in our studies, which concur with
previous work. We did not find T.sub.N cells in circulation,
target, or lymphoid organs in allogeneic recipients after day +7.
In contrast, we found T.sub.EM cells in circulation at day +14,
presumably en route from spleen to target organs. In our syngeneic
cohort, the number of T.sub.N cells increased over the course of
immune reconstitution, and was consistently higher than that of the
allogeneic cohort, even in thymectomized animals. This is another
feature of cGVHD that may have diagnostic importance. Given the
above points, we are developing a treatment strategy to selectively
inhibit in vivo CD4+ T.sub.EM subset expansion, including that
which occurs via T.sub.N conversion to T.sub.EM. As such, our
current work is focused on identifying metabolic differences
between T.sub.Reg, T.sub.N, and T.sub.EM cells early in the course
of allogeneic HSCT. Cell kinetics studies described herein have
formed the framework for this research, as they identified which
cell subsets should be pursued, their anatomic location, and
post-transplantation timing for further investigation.
[0123] When in vivo deuterated water labeling is conducted
post-HSCT, the DNA of rapidly proliferating cells, such as CD4+
T.sub.EM cells, becomes highly enriched with deuterium. As
illustrated by our kinetics studies, CD4+ T.sub.EM cells in
cGVHD-affected mice are preferentially enriched with deuterium to
31 15-20%, well above background total body water (TBW) enrichment
of .about.5%, which was implemented for kinetics studies. Combining
this deuterium enrichment difference and the contrasting
distribution of T cell subsets in cGVHD versus syngeneic
recipients, we hypothesized that target organs affected by cGVHD
could be visualized in vivo with deuterium chemical shift imaging
(CSI), also referred to as deuterium MRI (dMRI). We show that high
enrichment of deuterium within target organs does indeed allow
visualization of these organs by dMRI. As hypothesized, dMRI
provided a means by which cGVHD animals could be distinguished from
syngeneic counterparts. Thus, dMRI allows non-radioactive and
non-invasive diagnosis of cGVHD in our mouse model. Currently, no
imaging modalities for cGVHD are in use for cGVHD diagnosis in
patients. In clinical practice, biopsies are performed when
feasible, often limited by accessibility and morbidity concerns.
Hence, in vivo deuterium labeling followed by dMRI could serve as a
diagnostic clinical imaging modality for cGVHD, providing a
body-wide assessment of disease involvement.
[0124] Given that deuterium enrichment is tied to cell
proliferation, we demonstrate a first of many potential
translational applications for this non-invasive and
non-radioactive in vivo labeling-imaging approach. Deuterated water
labeling-imaging could facilitate non-invasive in vivo imaging of
many cell types, including neoplastic cells. As such, we have been
able to image mouse tumor cells labeled with deuterium in vitro,
and in vivo studies are planned. dMRI could provide an alternative
to current imaging modalities for cancer diagnosis and relapse
surveillance, such as computerized tomography (CT) and/or positron
emission tomography (PET), both of which involve radioactivity. In
addition, it is possible that dMRI could be used for in vivo
visualization of immunotherapeutic products post infusion, for
example, chimeric antigen receptor T cells (CAR T cells), tumor
infiltrating lymphocytes (TILs), and other adoptive
immunotherapies, if such products undergo labeling during
manufacture or expansion in culture prior to administration to a
patient.
[0125] In summary, the use of deuterated water labeling for dynamic
measurement of in vivo cell gain and cell loss kinetics and the
imaging of such cells in vivo via dMRI should illuminate
disease-specific pathophysiology, identify targets for therapeutic
interventions, and facilitate diagnosis of conditions characterized
by rapidly dividing cells.
[0126] Materials and Methods
[0127] Mice.
[0128] Three experimental cohorts were used: normal un-manipulated
mice, recipients of syngeneic HSCT, and recipients of allogeneic
HSCT. Female BALB/cAnNCr (H-2d) at 12-13 weeks of age served as
recipients/hosts. For syngeneic transplantation age-matched female
BALB/c mice served as donors. In a subset of experiments
thymectomized BALB/c mice served as recipients, and these
thymectomized animals were purchased. For allogeneic
transplantation age-matched female B10.D2-Hc1 H2-T18C/nSnJ (H-2d)
mice served as donors. Congenic experiments were conducted using
Thy1.1 B10.D2 and Thy1.1 Cg-FoxP3-GFP B10.D2 mice. BALB/c mice were
purchased from Charles River, Wilmington, Mass. B10.D2 mice were
purchased from Jackson Laboratories, Bar Harbor, Me. Congenic mice
were bred at the NCI Frederick, Md. breeding and holding facility.
All animal protocols were approved by the NCI Animal Care and Use
Committee.
[0129] Bone Marrow and Splenocyte Transplantation.
[0130] Recipient female mice were reconstituted with 8 million
un-fractionated splenocytes and 15 million bone marrow cells
injected via tail-vein on day 0, after 850 cGy TBI conditioning on
day -1, delivered in two divided doses 3 hours apart. Gentamicin
was added to injection buffer (100 .mu.g/ml). No additional
antibiotics were administered following transplantation. Congenic
experiments were conducted using an allogeneic graft comprised of
Thy1.1 Ly 9.2 spleen cells and Thy 1.2 Ly 9.2 T cell-depleted
marrow transplanted into Thy1.2, Ly 9.1 hosts. Mice received
regular drinking water and food until .sup.2H.sub.2O water labeling
commenced. cGVHD scoring was performed twice weekly using
previously described clinical scoring system. Animals were weighed
weekly and on the day of euthanasia for experiments involving
dermal and peripheral blood flow cytometry.
[0131] .sup.2H.sub.2O (Deuterated Water) Labeling.
[0132] Deuterated water labeling was performed according to
previously published protocol. Briefly, deuterated water was
provided in drinking water after an initial intravenous bolus for
specified labeling periods, during which newly synthesized cells
incorporate deuterium into DNA base pairs. Incorporation of the
label occurs through the de novo synthesis of nucleosides.
Sequential labeling and pulse-chase experiments, allow quantitative
measurements of cell gain and cell loss, respectively, in an organ
or tissue from which the cells are extracted. For cell gain
kinetics, the fraction of newly synthesized cells during each 7-day
labeling period was calculated by dividing dA M+1 for the
population of interest by dA M+1 for a reference fully turned over
population (unfractionated bone marrow). Unfractionated bone marrow
was collected for each experimental cohort as a pooled sample at
each time point. De-labeling kinetics were obtained by measuring
day +14 dA M+1 with a preceding 7-day labeling period. Thereafter,
label administration ceased and mice received regular drinking
water. Measurements for dA+M1 were obtained at day +21, +28 and +35
for spleen de-labeling experiments and day +21 and +28 for liver
de-labeling experiments. The enrichment values were then plotted
over time. Formulas for de-labeling curves were obtained in
Microsoft Excel for Mac 2011 with linear, logarithmic or polynomial
fitting.
[0133] Urine Sample Collection.
[0134] For each mouse that underwent dMRI, .about.50 ul of urine
was collected prior to imaging. Urine collection was facilitated by
placing the mouse on a strip of parafilm, then gently massaging the
bilateral flanks. Upon spontaneous passage of urine onto parafilm,
the urine was transferred into 1.5-ml Eppendorf with a transfer
pipette. The urine samples were stored at -20.degree. C. until
total body water (TBW) deuterium enrichment analysis on GC-MS/MS
was performed.
[0135] Organ Collection and Preparation.
[0136] Integument.
[0137] The harvesting of lymphocytes from flank sections was
performed according to previously published methodology. Briefly,
dorsal skin was harvested and subcutaneous adipose tissue was
removed. One cm.sup.2 sections underwent enzymatic digest in
Liberase TL (Roche) for 2 hours at 37.degree. C. and 5% CO.sub.2.
Digested skin sections were then loaded into medicon cartridges and
mechanically ground on the Medimachine System (Becton Dickinson).
While lymphocytes were extracted from one cm.sup.2 sections from
each mouse, total body surface area was calculated for each mouse
based on weight (BSA=k mass.sup.0.667, where k is the Meeh constant
empirically determined for each species) allowing whole skin
lymphocyte content to be estimated.
[0138] Liver.
[0139] Circulating non-parenchymal blood was flushed out of the
liver prior to organ harvest by intra-cardiac injection of 20 ml
PBS with outflow through the cut portal vein. Gallbladder was
removed prior to processing. Tissue was mechanically disrupted and
sequentially filtered through 100 .mu.m, 70 .mu.m, and finally 40
.mu.m filters. ACK lysis was used prior to re-suspending cells for
counting.
[0140] Small Intestine.
[0141] The small intestines were harvested and processed per
previously described protocol. Briefly, the small intestine was cut
proximally at the pyloric junction, then drawn out of the
peritoneal cavity. Adipose tissue was manually removed. Another cut
was made at the cecal junction and the small intestine was then
removed from the carcass. Intestinal tissue was placed into medium
containing 3% fetal calf serum (FCS) in RPMI (3% media) on ice.
Peyer's patches were then removed and processed separately (per
Lymphoid organs preparative procedure). The small intestine was cut
longitudinally and fecal matter was manually removed. Residual
fecal material was rinsed off with 3% media, followed by a rinse in
Hank's Balanced Salt Solution (HBSS). The tissue was then cut into
1-cm sections and placed into solution containing 0.145 mg/ml DTT
(dichlorodiphenyltrichloroethane) in 3% media and incubated for 20
minutes at 37.degree. C. with continuous mixing (magnetic stirrer
mixing at .about.800 RPM). Following incubation, the contents were
filtered through a stainless steel strainer. The suspension
containing the intraepithelial lymphocytes (IEL) was placed on ice.
The remaining tissue (on the strainer) was transferred into 0.5M
EDTA solution, then vigorously shaken. Following this step, the
solution was passed over the strainer and the liquid portion was
combined with the rest of IEL (subsequent preparation steps are
described under Isolation of IEL section).
[0142] Isolation of Lamina Propria (LP) Lymphocytes.
[0143] The remaining intestinal sections were placed into solution
containing 0.1 mg/ml Liberase TL (Roche) and 0.1 mg/mL DNase I
(Sigma-Aldrich) and were finely minced. The mixture was then
incubated for 30 min @ 37.degree. C. with continuous stirring at
.about.800 RPM. The solution was placed on ice and 0.1 mg/mL DNase
was added. The contents were then passed over a 70 .mu.m filter.
The remnant intestinal pieces were crushed on the filter and rinsed
with 0.1 mg/ml DNase solution. The solution was then spun for 5
minutes at 4.degree. C. and 1,300 RPM. The cell pellet was
re-suspended in 3% media and filtered over 40 .mu.m filter. Spin
step was repeated and the cells were re-suspended in 10% FCS in
RPMI media for cell counting.
[0144] Isolation of Intra-Epithelial Lymphocytes (IEL).
[0145] The solution containing IEL was spun for 7 minutes at
4.degree. C. and 1,400 RPM. The pellet was re-suspended in 3%
media, then filtered over 40 .mu.m filter, followed by another spin
step. The pellet was then re-suspended in 30% Percoll solution (GE
Healthcare) and spun for 20 minutes at room temperature and 1,600
RPM. The cell pellet was re-suspended in 0% FCS media and spun for
7 minutes at 4.degree. C. and 1,400 RPM. The remaining cell pellet
was re-suspended in 10% FCS RPMI media for cell counting.
[0146] Lymphoid Organs.
[0147] Single cell suspensions were obtained by mechanically
disrupting lymphoid organs (thymus, spleen, and lymph nodes,
including submandibular, axillary, inguinal, mesenteric, and
Peyer's patches), then filtering through 70 .mu.m filters. ACK
lysis was performed on spleen samples to remove red blood cells
prior to cell counting.
[0148] Peripheral Blood.
[0149] Blood was collected by orbital sinus canulation with
heparinized glass tubes and placed on ice. Total volume of sample
was recorded. Samples were then spun for 5 minutes at 4.degree. C.
and 5,000 RPM. Serum was removed and the samples underwent two ACK
lysis steps. Cells were then re-suspended in media for counting.
Total blood volume for each mouse based on 7% body weight was used
to estimate total lymphocytes present in entire blood volume for
each mouse by extrapolating lymphocyte numbers obtained from
experimental samples of known collection volume.
[0150] Staining for Flow Cytometry and Fluorescence-Activated Cell
Sorting.
[0151] Cells were counted using Nexcelom Cellometer Auto T4 (Life
Technologies, Grand Island, N.Y.) and Trypan Blue 0.4% (Lonza). One
to two million cells was aliquoted for flow cytometry staining. For
sorting, samples from multiple mice were pooled for each cohort
(normal mice, syngeneic recipients and allogeneic recipients).
Surface antibody staining was performed on single-cell suspensions.
The following antibodies were purchased from eBioscience, BD
Biosciences, BioLegend, or Invitrogen: anti-Active Caspase-3
(559341), anti-mouse CD4 (GK1.5), anti-mouse CD8a (5H10, 53-6.7),
anti-mouse CD8.beta., anti-mouse CD16/CD32 Fc block (2.4G2),
anti-mouse CD25 (PC61.5), anti-mouse CD69 (H1.2F3),
anti-mouse/human CD44 (IM7), anti-CD90.2 (Thy1.2) (53-2.1),
anti-mouse CD197/CCR7 (4B12), anti-mouse/rat Foxp3 (FJK-165),
anti-mouse CD229.1 (Ly9.1) (3007), Streptavidin Pacific Blue
(S-11222), anti-mouse .gamma..delta. TCR (eBioGL3), anti-TCR .beta.
(H57-597). LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen)
was used to exclude dead cells. mCD1d (PBS-57) Tetramer was
obtained from NIH Tetramer Core Facility (Atlanta, Ga.) for
staining liver parenchyma, to allow exclusion of NKT cells from T
cells for FACS and flow cytometry phenotyping. The cells were fixed
with eBioscience Fixation/Permeabilization reagents, and then
intra-cellular staining for Foxp3 was performed overnight at
4.degree. C. Pooled samples for each cohort underwent
fluoresce-activated cell sorting (FACS) using Becton-Dickinson (BD)
Influx (San Jose, Calif.), with 95% purity. Individual mouse sample
flow cytometry measurements were obtained on BD LSR II and
collected data were analyzed using FlowJo 9.7.6 Software (Ashland,
Oreg.). FACS-purified samples were collected into PBS buffer
containing 2% bovine serum albumin. The samples were spun for 10
minutes at 4.degree. C. and 10,000 RPM. Supernatant was removed and
remaining cell pellets were stored at -80.degree. C. until DNA
extraction.
[0152] DNA Extraction.
[0153] DNA extraction from non-fixed cells was performed on the
Promega Maxwell 16 system (Madison, Wis.) as previously described.
For DNA extraction from sorted fixed cells, an EpiSonic.TM.1100
Sonication System (Epigentek, Farmingdale, N.Y.) was used.
[0154] GC-MS/MS Analysis
[0155] dA Enrichment Measurements.
[0156] Quantitative determination of deoxyadenosine (dA), its
isotopologue (dA M+1) and the internal standard (dA M+5) was
measured using validated GC-MS/MS methodology. Briefly, DNA
extracted from FACS purified T cell subsets was hydrolyzed to its
base pairs using EpiQuick DNA Hydrolysis Kit (Epigentek). The base
pairs were then purified and concentrated using solid phase
extraction (SPE). The SPE extracts were dried under vacuum, and
MethElute.TM. (methylation reagent) was added to the residue and
mixed thoroughly. The Agilent 7890A GC, LTM series II fast GC
module, 7000A GC-MS triple quadrupole, and 7693 auto sampler (Santa
Clara, Calif.) were then used. Upon injection into the GC, the
derivatized base pairs were separated using low thermal mass fast
gas chromatography. Calibration standards of dA, dA M+1 and the
internal standard (dA M+5) were used for quantitative mass
spectrometry, utilizing positive chemical ionization and the MRM
mode of MS detection.
[0157] Total Body Water (TBW) Enrichment Measurements.
[0158] For measuring .sup.2H.sub.2O water levels in TBW (e.g.
urine), we developed a simple and quantitative headspace-GC-NCI-MS
method (publication pending). Briefly, the method utilizes a rapid
gas phase isotopic exchange of the .sup.1H:.sup.2H moiety between
.sup.2H.sub.2O water in TBW and the acetone solvent used for
isotopic exchange. The method requires 25 .mu.L of TBW sample, i.e.
urine, demonstrates a linear relationship from 2-40%
(v/v).sup.2H.sub.2O in TBW, and has a total analysis time of less
than 10 min
[0159] Histopathology.
[0160] Thymus, lymph nodes, spleen, liver, stomach, small
intestine, colon, skin, lung, and bone marrow (sternum) were
extracted from mice immediately following euthanasia and placed
into 4% w/v formaldehyde. Tissue cassettes were then sent to
Histoserv, Inc. (Germantown, Md.) for sectioning and H & E
staining. H & E slides were evaluated for cGVHD histological
grading by Dr. ME. Adobe Photoshop Elements 8.0 was used to acquire
images of the H & E figures obtained via Olympus DP12 camera
visualized through an Olympus BX41 microscope, magnification as
noted on each figure.
[0161] Proton and Deuterium MRI.
[0162] All magnetic resonance imaging (MRI) experiments were
performed on a 9.4 Tesla magnet equipped with a Bruker Advance III
MRI console (Bruker-Biospin, Billerica, Mass.) Immediately
following euthanasia, each mouse was wrapped in plastic and taped
onto a plastic cradle in a flat, level position. A 5-mm diameter
tube (phantom) containing 5% .sup.2H.sub.2O in ddH.sub.2O with 0.1%
sodium azide (preservative) was placed adjacent to the mouse as a
reference and calibration standard. The cradle was centered in the
MRI probe described above, and then placed in the magnet. Following
acquisition of a set of standard locator images, a set of three
coronal planes were prescribed covering the spleen and liver.
Reference images of these regions were acquired using the MSME
sequence and the following parameters: Field of View=40.times.40
mm, slice thickness=1 mm, TR/TE=1000/14 ms, and the matrix of
256.times.256. Subsequently, the same planes were imaged using the
deuterium chemical shift imaging (CSI) sequence with the following
parameters: Field of View=40.times.40 mm, slice thickness=3 mm,
TR/TE=398/1.6 ms, spatial matrix=128.times.64, and spectral
matrix=512. The excitation flip angle was adjusted to meet an Ernst
angle condition. All imaging data were analyzed using custom code
written in Python. Following CSI reconstruction, regions of
interest were placed onto the reference sample, the spleen (when
visible), the liver, and muscle. The average intensity and the
standard deviation of the intensity was measured and tabulated for
each region on the CSI and reference images. Anatomical regions
were normalized using the reference (5% .sup.2H.sub.2O phantom)
intensity to compensate for experimental variation.
[0163] .sup.1H-.sup.2H, Proton-Deuterium, Coil for MRI.
[0164] The schematic for the components of the proton-deuterium
coil is provided in FIGS. 29A-B.
[0165] Statistical Analysis.
[0166] Data for experimental cohorts were graphed with Prism 6.0
(GraphPad Software, Inc.) or Microsoft Excel for Mac 2011. Error
bars on bar graphs represented standard error of the mean (SEM).
Data for experimental cohorts were compared with Microsoft Excel
for Mac 2011 or Minitab 16.2.4 (State College, Pa.) using
two-sample, two-tailed, unequal variance t-test. P values were
assigned a single asterisk (*) when they were <0.05, and those
marked with ** were <0.01.
[0167] It should be understood from the aforementioned descriptions
that while particular embodiments have been illustrated and
described, various modifications can be made thereto without
departing from the spirit and scope of the invention as will be
apparent to those skilled in the art. Such changes and
modifications are within the scope and teachings of this invention
as defined in the claims appended hereto.
* * * * *